Methods and compositions for stimulating T-lymphocytes

ABSTRACT

Disclosed are methods, compositions, antibodies, and therapeutic kits for use in stimulating cytotoxic T-lymphocytes and generating immune responses against epitopes of protooncogenes. Novel peptides are described which have been shown to stimulate cytotoxic T-lymphocytes, and act as antigens in generation of oncogenic epitope-recognizing antibodies. Methods are disclosed for use in treating various proliferative disorders, and diagnosing HER-2/neu-containing cells; also disclosed are therapeutic kits useful in the treatment of cancer and production of potential anti-cancer vaccines.

[0001] The United States government owns rights to the present inventionpursuant to Grants CA 57293 and CA 16672 from the National CancerInstitute.

BACKGROUND OF THE INVENTION

[0002] A. Field of the Invention

[0003] The present invention relates generally to the field of molecularbiology, and particularly to the area of natural and synthetic peptides.More particularly, the invention discloses HER-2/neu peptide, DNAsegment, antibody compositions. Various methods for making and usingthese compositions are disclosed, such as, for example, the use ofpeptides and antibodies in various pharmacological and immunologicalapplications, including the stimulation of cytotoxic T-lymphocytes andcancer therapies.

[0004] B. Description of the Related Art

[0005] 1. HER-2/neu Proto-oncogene

[0006] The HER-2/neu proto-oncogene (HER-2) encodes a transmembraneprotein whose expression is enhanced in a number of breast and ovariantumors and correlates with tumor aggressiveness. Because of itsexpression on normal epithelial cells, HER-2 can be defined as atumor-associated antigen (Ag) and may be of interest as a target of atherapeutic anti-tumor T-cell response. A CD3⁺CD8⁺CD4⁻ line isolatedfrom cell cultures have been shown to lyse HLA-A2⁺, HER-s⁺ ovariantumors but not natural killer (NK) target K562 cells, and showedsignificantly higher lysis of HER-2^(high) than of HER-2^(low) ovariantumors. Some inhibition of lysis was inhibited by HER-2 peptide-pulsedHLA-A2⁺ targets, suggesting that some epitopes may be present on tumorcells associated with HLA-A2.

[0007] 2. Tumor-reactive T-cells

[0008] Tumor reactive T-cells have been reported to mediate therapeuticresponses against human cancers (Rosenberg et al., 1988). In certaininstances, in human immunotherapy trials with tumor infiltratinglymphocytes (TIL) or tumor vaccines, these responses correlated eitherwith in vitro cytotoxicity levels against autologous tumors (Aebersoldet al., 1991) or with expression of certain HLA-A,B,C gene products(Marincola et al., 1992). Recent studies (Ioannides et al., 1992) haveproposed that in addition to virally encoded and mutated oncogenes,overexpressed self-proteins may elicit some degree of tumor-reactivecytotoxic T-lymphocytes (CTLs) in patients with various malignancies(Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al., 1993;Jerome et al., 1991). Autologous tumor reactive CTLs can be generatedfrom lymphocytes infiltrating ovarian malignant ascites (Ioannides etal., 1991), and overexpressed proteins such as HER-2 may be targets forCTL recognition (Ioannides et al., 1992).

[0009] Information on epitopes of self-proteins recognized in thecontext of MHC Class I molecules remain limited, despite a few attemptsto identify epitopes capable of in vitro priming and Ag-specificexpansion of human CTLs. For example, peptide epitopes have beenproposed which are likely candidates for binding on particular MHC ClassI Ag (Falk et al., 1991), and some studies have attempted to definepeptide epitopes which bind MHC Class I antigens.

[0010] Short synthetic peptides have been used either as target antigensfor epitope mapping or for induction of in vitro primary and secondaryCTL responses to viral and parasitic Ags (Bednarek et al., 1991; Gammonet al., 1992; Schmidt et al., 1992; Kos and Müllbacher, 1992; Hill etal., 1992). Unfortunately, these studies failed to show the ability ofproto-oncogene peptide analogs to stimulate in vitro human CTLs to lysetumors endogenously expressing these antigens.

[0011] 3. Synthetic Peptides and T-cell Epitope Mapping

[0012] Synthetic peptides have been shown to be a useful tool for T-cellepitope mapping. However in vivo and in vitro priming of specific CTLshas encountered difficulties (Alexander et al., 1991; Schild et al.,1991; Carbone et al., 1988). It is generally considered that in vitroCTL priming cannot necessarily be achieved with peptide alone, and infact, a high antigen density is thought to be required for peptidepriming (Alexander et al., 1991). Even in the limited instances whenspecific priming was achieved, APC or stimulators were also required athigh densities (Alexander et al., 1991).

[0013] It is not clear when CTL induction by HER-2 peptides in vitro wasobserved whether this reflects secondary activation of CTL specific for,or cross-reacting with, the Ag of interest. Whether or not thiscross-reactivity can constitute the foundation for development of an invitro CTL response to tumor remains to be determined.

[0014] Therefore, what is lacking in the prior art are universalepitopes which are both immunodominant and CTL-stimulating. Moreover,methods for the use of such CTL-stimulating peptides would be mostdesirable in the treatment of human cancers, particularly of breast andovarian etiology, and the development of cancer vaccines. Identifyinguniversal oncoprotein epitopes would permit not only an increasedunderstanding of tumor immunity and autoimmunity in humans, but wouldalso open the door to the design of novel therapeutic strategies forproliferative cell disorders such as human cancers, and particularlybreast and ovarian cancers.

SUMMARY OF THE INVENTION

[0015] The present invention seeks to overcome these and other inherentdeficiencies in the prior art by providing the identification of nativeand synthetic proteins or peptides derived from the HER-2/neuproto-oncogene gene product, and methods for their use in stimulatingcytotoxic T-lymphocytes.

[0016] These selected “universal” immunodominant epitopic peptides, andtheir synthetically-optimized derivatives are envisioned to be useful inthe development of tumor vaccines, and anti-cancer therapeutics.Pharmaceutical reagents resulting from these novel peptides and the DNAsegments which encode them will also likely prove useful as testreagents for the detection of HER-2/neu-related polypeptides, facilitatethe production of anti-peptide antibodies specific to a range ofHER-2/neu-related polypeptides, and result in the stimulation andproduction of cytotoxic T-lymphocytes specific for a variety ofproliferative disorders including human cancer.

[0017] Synthetic peptide analogs can be used to define CTL epitopesrecognized by tumor reactive T-cells and to stimulate in vitropeptide-specific CTLs. Such CTLs can be further evaluated forrecognition of targets endogenously expressing the particular antigen(Ag) and for Ag-specific adoptive therapy.

[0018] Disclosed herein are compositions and methods for their makingand use in development of anti-cancer vaccines. The generation in vitroof HLA-A2-restricted CTLs using HER-2 synthetic peptide analogs asimmunogens, and peripheral blood mononuclear cells (PBMC) from healthyvolunteers as responder cells is also described. Lysis with isolatedCD8⁺ T-cells from these CTL cultures was observed using both HER-2peptide-pulsed HLA-A2 from these CTL cultures was observed using bothHER-2 peptide-pulsed HLA-A2 transfectants and HLA-A2⁺ ovarian tumorsexpressing high levels of HER-2 as targets.

[0019] Another aspect of the invention is the development andmaintenance in long-term culture a CD3⁺CD8⁺CD4⁻ line by restimulationwith HER-2 peptide-pulsed autologous PBMC. This line lysed HLA-A2⁺,HER-2^(high) ovarian tumors, but not HLA-A2⁺, HER-2^(low) ovariantumors. Tumor lysis was inhibited by HER-2 peptide-pulsed HLA-A2⁺transfectants, demonstrating that epitopes either similar orcross-reactive with the ones recognized by CTLs on the peptide used asimmunogen in vitro are present on the tumor cells. These CTL showedlower lysis of targets pulsed with unrelated peptides (analogs of Muc-1core peptide where HLA-A2 anchors were introduced).

[0020] A novel approach to developing tumor reactive CTLs is disclosedwhich focuses on a target Ag expressed on the tumor of interest andidentifying CTLs induced in vivo or developed in vitro that recognizethis target Ag. In tumor cells the level of expression of a particularprotein may be 10²-10³ fold higher than in normal tissue.

[0021] The inventors expect that a number of target T-cell Ags on humantumors may be derived from proteins that are expressed at low levels innormal cells, and at significantly higher concentration in tumor cells,such as overexpressed proto-oncogene products (Ioannides et al., 1992).The rationale for this hypothesis is: first, peptides from self-proteinswhich fulfill the criteria of MHC allele-specific motifs should becapable of binding to the Ag binding pockets in the MHC class I heavychain; and second, positive and negative selection of T-cell repertoiremay result in elimination or tolerization of high-affinity self-reactiveCTLs (Parmianai, 1993), although such peptide-MHC complexes should havelower affinity for the TCR than a de novo expressed epitope from aself-protein (as a consequence either of mutations creating HLA-anchorsor modifying the core recognized by the TCR), their presence in highconcentration may engage a large number of TCR.

[0022] The HER-2/neu proto-oncogene was identified because it isoverexpressed (in certain instances by several hundred fold) in a numberof breast and ovarian tumors (Slamon et al., 1989). Moreover, it wasfound that several CTL-TAL lines isolated from ovarian malignant ascitescould lyse autologous ovarian tumors.

[0023] Surprisingly, the inventors also discovered that this lysis couldalso be effectively inhibited by natural and synthetic peptide analogsof HER-2. These results suggested that these novel peptides acted asepitopes that were either derived from an endogenously-processed HER-2peptide, mimicked, or cross-reacted with a peptide of related sequencederived from another protein.

[0024] Novel synthetic peptide compositions have also been developedwhich correspond to the HER-2:968-981 and 971-979 regions. Thecompositions disclosed herein, were found to stimulate in vitro PBMCsfrom healthy HLA-A2⁺ human volunteers (Fisk et al., 1994), and CTLs(induced by peptide stimulation) consequently lysed tumorsoverexpressing HER-2 (Fisk et al., 1994). These studies demonstratedthat these CTLs can effectively recognize the epitope peptides of thepresent invention, and that these HER-2-derived peptides can stimulatein vitro PBMCs to induce peptide reactive CTLs.

[0025] This possibility may be particularly relevant for induction of Agand tumor-specific CTLs because peripheral T-cells that can recognizesuch peptides from non-mutated self proteins are those that have eitherescaped elimination or may have become tolerant to one or more of theseantigenic epitopes due to low affinity TCR-MHC interactions (Ioannideset al., 1992; Parmiani, 1993).

[0026] Other aspects of this invention include the identification ofcandidate HER-2-derived T-cell epitopes based on the presence of anchorsfor HLA-A2, the analysis of these peptides to affect the conformation ofHLA-A2 as an indication of peptide binding, and finally, thedemonstration that these peptides can stimulate in vitro peptidereactive CTLs from human HLA-A2⁺ PBMC.

[0027] Methods are described herein for stimulation of CTLs (andconsequently, production of an immune response) employing the novelcompositions disclosed herein. In vitro induction of cellular responsesto the peptides of the present invention by PBMC from healthy HLA-A2⁺volunteers demonstrated their ability to stimulate and/or restimulatepre-existing T-cell responses to HER-2. The peptides inducedproliferative responses in one of four donors tested and CTL responses(one of three peptides tested in two of three donors), and may be usedto induce tumor-reactive T-cells in vitro and in vivo through eitherpeptide-, lipopeptide-, or cell-mediated methods. These peptidestherefore find utility in both generating an immune response, andserving as antigens in the preparation of peptide-specific antibodies.

[0028] The peptides of this invention also may be used in embodimentsinvolving treatment, diagnosis, and identification of proliferative celldisorders such as cancer, and particularly cancers such as, inter alia,breast and ovarian tumors. Methods of identification ofHER-2/neu-containing cells, and also neu-related proto-oncogene andoncogene products are also disclosed.

[0029] Cancer treatment methods, including vaccine development areanother aspect of the present invention. Additionally, a variety of invitro and in vivo assay protocols are facilitated as a result of thenovel compositions disclosed herein. In addition to stimulating CTLs,and generating an immune response in an animal, and particularly in ahuman, the peptides may also be used as immunogens to generateanti-peptide antibodies, which themselves have many uses, not least ofwhich is the detection of oncogene-containing cells (e.g., detection ofHER-2/neu, related oncogenic polypeptides, or peptide fragments thereof,in diagnostic tests and kits based upon immunological binding assays).

[0030] Also, since the peptides of the invention bind to T-cells, theymay be employed in assays to identify T-cells, and particularly CTLs,for example, to assess the immunological capacity of a given individualor animal, or even to purify CTLs themselves. Such methods could utilizeradioactively- or enzymatically-labeled peptides or anti-peptideantibodies, such as those described herein.

[0031] Therefore, one contemplated use for the described peptidesconcerns their use in methods for detecting the presence of T-cellswithin a sample. These methods include contacting a sample suspected ofcontaining T-cells with a peptide or composition in accordance with thepresent invention under conditions effective to allow the peptide(s) toform a complex with T-cells of the sample. One then detects the presenceof the complex by detecting the presence of the peptide(s) within thecomplex, e.g., by either originally using radiolabeled peptides or bysubsequently employing anti-peptide antibodies and standard secondaryantibody detection techniques.

[0032] Preferred peptides of the present invention will likely be fromabout 6 to about 20 amino acids, in length, with peptides of from 7 toabout 15 amino acids in length being even more preferred. Most preferredare peptides having lengths of from about 8 to about 10 amino acids inlength, with nonameric and decameric peptides being most preferred.These peptides may include one or more D-amino acids, or may even beentirely composed of D-amino acids, and may, of course, containadditional elements, as desired for stability or even for targetingpurposes.

[0033] The peptides, or multimers thereof, may be dispersed in any oneof the many pharmacologically-acceptable vehicles known in the art andparticularly exemplified herein. As such, the peptides may beencapsulated within liposomes or incorporated in a biocompatible coatingdesigned for slow-release. The preparation and use of appropriatetherapeutic formulations will be known to those of skill in the art inlight of the present disclosure. The peptides may also be used as partof a prophylactic regimen designed to prevent, or protect against,possible cancer progression and/or metastasis and may thus be formulatedas a vaccine, particularly as a method of stimulating anti-tumor CTLs.

[0034] The present invention also provides methods for identifyingHER-2/neu and related proto-oncogene products, which methods comprisecontacting the cells suspected of containing such polypeptides with animmunologically effective amount of a composition comprising one or morespecific anti-peptide antibodies disclosed herein. Peptides that includethe amino acid sequence of any of SEQ ID NO: 1 through SEQ ID NO: 29 andtheir derivatives will be preferred for use in generating suchanti-CTL-stimulating peptide antibodies.

[0035] The invention thus also provides compositions, includingpeptides, peptide multimers, and pharmaceutical compositions derivedtherefrom, that contain one or more peptides of from 8 to about 20 aminoacids in length that include within their sequence the peptide sequenceidentified by the formula: AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-AA₈; where AA₁ isLeu, Met, Ile, or Val; AA₂ is any amino acid; AA₃ is any amino acid; AA₄is Ser, Glu, Thr, or Tyr; AA₅ is any amino acid; AA₆ is any amino acid;AA₇ is any amino acid; and AA₈ is Val, Leu, Met, Ile, or Cys. Thesepeptides are submitted to be capable of stimulating CTLs and producingan immune response in vitro and in vivo.

[0036] Another aspect of the present invention concerns the use of theamino acid sequences disclosed herein in the determination of molecularweights of low-molecular-weight polypeptides. These peptides represent asignificant improvement over commercially-available protein standards inthis area owing to their small size, and the presence of knownnonapeptide motifs. Commercially-available standards typically have arange of 3,000 to 200,000 Da, and as such, are not useful in thecharacterization of proteins having molecular weights of about 300 toabout 3,000 Da using either conventional or gradient SDS-PAGE.

[0037] In a similar fashion, the peptides, and more particularly peptideoligomers, of the present invention are readily employed as standards inthe identification of small molecular-weight polypeptides usingchromatographic separation. In preferred embodiments, paperchromatography is utilized and proteins are subsequently visualizedafter reaction with ninhydrin. More preferred is the use of thin-layerchromatography in either one or two dimensions.

[0038] The use of the peptides and peptide motifs of the presentinvention is also contemplated for the calibration and standardizationof chromatographic columns used in the separation oflow-molecular-weight polypeptides. These peptides, and multimersthereof, find important use in the calibration oflow-molecular-weight-range columns. Such molecular sieve (or gelfiltration) chromatography columns may include a filtration mediumhaving the capacity to fractionate any protein of interest and thepeptides of the present invention. Preferred chromatographic media wouldinclude any gel filtration medium having a molecular fractionation rangesuitable for the particular protein of interest. Preferred media wouldinclude the G-50 or G-25 Sephadex® resins which have an approximatefractionation range of 1,500-30,000 and 100-5,000 Da, respectively. Amore preferred medium would be either the G-10 or G-15 Sephadex® resinswhich have an approximate fractionation range of 0-700 and 0-1500 Da,respectively.

[0039] Peptides of the present invention comprising aromatic amino acidsand multimers thereof may also be used as protein concentrationstandards in reactions employing either the Folin reagent (Lowry et al.,1951), the biuret reaction (Coakley and James, 1978) or the bicinconinicacid assay (Pierce Chemical Corp., Rockford, Ill.). Peptides andmultimers thereof lacking aromatic amino acids may also be used asprotein concentration standards in the latter two reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1A. Lysis of C1R:A2 cells after sensitization with peptidesC43 (HER-2:968-981) (▪) and C84 (HER-2:921-979(Val) (□) or in theabsence of exogenously added peptides (0) by CTL cultures induced invitro with C43 and C84 peptides. Headings indicate: donor number (e.g.,51), number of stimulations with peptide (e.g., 2×/3×), and the peptideused for stimulation.

[0041] Donor 51 PBMC were tested 3 weeks after the second stimulationwith the C43 peptide (total 5 weeks in culture). The studies wereperformed in triplicate. The differences between individualdeterminations were less than 10%. The differences between HER-2 peptideand control targets recognition are significant in 20-hr assays (P<0.003for C43 and P<0.027 for C84) and are not significant (P<0.10) in 4-hrassays. The effector to target ratio was 10:1.

[0042]FIG. 1B. Lysis of C1R:A2 cells after sensitization with peptidesC43 (HER-2:968-981) (▪) and C84 (HER-2:921-979(Val) (□) or in theabsence of exogenously added peptides (0) by CTL cultures induced invitro with C43 and C84 peptides. Headings indicate: donor number (e.g.,51), number of stimulations with peptide (e.g., 2×/3×), and the peptideused for stimulation. Shown is the donor 51 PBMC were stimulated twotimes with the C84 peptide and tested 3 weeks after.

[0043]FIG. 1C. Lysis of C1R:A2 cells after sensitization with peptidesC43 (HER-2:968-981) (▪) and C84 (HER-2:921-979(Val) (□) or in theabsence of exogenously added peptides (0) by CTL cultures induced invitro with C43 and C84 peptides. Headings indicate: donor number (e.g.,51), number of stimulations with peptide (e.g., 2×/3×), and the peptideused for stimulation. Shown is the donor 41 PBMC stimulated two timeswith C84.

[0044]FIG. 2A. Ag specificity of the 41.CD8⁺ CTL line. C1R:A2 cells werepre-pulsed with either HER-2 peptides or control MUC-1 peptides beforebeing incubated with effectors. The effector to target ratio was 10:1.C1R:A1 and C1R:A3 targets were pre-pulsed with the same peptides in thesame conditions as C1R:A2 cells. Results for C1R:A1 and C1R:A3 show thedifference between specific lysis of targets preincubated with peptidesand control C1R:A1 and C1R:A3 targets. Specific lysis of control C1R:A1and C1R:A3 cells was less than 10% at the same E:T ratio. Shown in FIG.2A are results after 4 hrs' incubation.

[0045]FIG. 2B. Ag specificity of the 41.CD8⁺ CTL line. C1R:A2 cells werepre-pulsed with either HER-2 peptides or control MUC-1 peptides beforebeing incubated with effectors. The effector to target ratio was 10:1.C1R:A1 and C1R:A3 targets were pre-pulsed with the same peptides in thesame conditions as C1R:A2 cells. Results for C1R:A1 and C1R:A3 show thedifference between specific lysis of targets preincubated with peptidesand control C1R:A1 and C1R:A3 targets. Specific lysis of control C1R:A1and C1R:A3 cells was less than 10% at the same E:T ratio. Shown in FIG.2B are results after 20 hrs incubation.

[0046]FIG. 3A. Lysis of fresh isolated ovarian tumor OVA-16 (HLA-A2⁺,HER-2^(high)) cells by the donor 41 CD8⁺ cell line. Target lysis wasdetermined in 5-hr (□) and 20-hr (▪) assays in the same study againstboth targets.

[0047]FIG. 3B. Lysis of fresh isolated ovarian tumor K562 cells by thedonor 41 CD8⁺ cell line. Target lysis was determined in 5-hr (□) and20-hr (▪) assays in the same study against both targets.

[0048]FIG. 3C. Lysis by 41.CD8⁺ CTL of HLA-A2⁺ HER-2^(high), HER-2^(low)ovarian tumors and HLA-A3⁺ HER-2^(high) (SKOV3) ovarian and HLA-A11⁺HER-2^(high) (SKBr3) breast tumor lines. C1R:A2 and XX Cr cells werenegative control targets.

[0049]FIG. 4A. Target specificity of the 41.CD8⁺ CTL were tested for theability to lyse ⁵¹Cr-labeled OVA-16 at an E:T ratio of 10:1. C1R:A2cells (A2.1) were incubated with synthetic peptides (D125, C43, C85),washed, and used in cold target inhibition studies at a cold:hot ratioof 2:1. Cytotoxicity studies were performed for 5 hr. Results representthe mean of three determinations. The variability between samples wasless than 10%. The differences between determination are statisticallysignificant (P<0.03) as determined by Student's t test. Percentageinhibition is indicated in parentheses.

[0050]FIG. 4B. Target specificity of the 41.CD8⁺ CTL were tested for theability to lyse ⁵¹Cr-labeled OVA-16 at an E:T ratio of 10:1. C1R:A2cells (A2.1) were incubated with synthetic peptides (D125, C43, C85),washed, and used in cold target inhibition studies at a cold:hot ratioof 2:1. Cytotoxicity studies were performed for 20 hr. Results representthe mean of three determinations. The variability between samples wasless than 10%. The differences between determination are statisticallysignificant (P<0.03) as determined by Student's t test.

[0051] Percentage inhibition is indicated in parentheses.

[0052]FIG. 5A. Effects of HER-2 peptides on reactivity of MA2.1 mAb withT2 cells. Fluorescence analysis and determination of FL1 were performedas described (Stauss et al., 1992). Peptides were added to T2 cells at50 μg/ml (final concentration). After overnight culture, in IMDM-FCS,cells were washed and the levels of HLA-A2 expression were determinedusing HLA-A2 specific mAb. Control indicates that no exogenous peptideswas added in the T2 cultures. D98, D160, and D169 are control peptideswhich do not contain HLA-A2 anchors in correct positions.

[0053]FIG. 5B. Effects of HER-2 peptides on reactivity of BB7.2 mAb withT2 cells. Studies were performed as described in the legend to FIG. 5A.

[0054]FIG. 5C. Effects of HER-2 peptides on reactivity of MA2.1 mAb withT2 cells. Studies were performed as described in the legend to FIG. 5A.

[0055]FIG. 5D. Effects of HER-2 peptides on reactivity of BB7.2 mAb withT2 cells. Studies were performed as described in the legend to FIG. 5A.

[0056]FIG. 6. Effects of Folate Binding Protein (FBP) peptides onreactivity of MA2.1 mAb with T2 cells. Experimental conditions asdescribed in the legend to FIG. 5A. Control column indicates that T2cells were cultured in the absence of peptide.

[0057]FIG. 7A. Surface phenotype of T-cells from PBMC culturesstimulated with HER-2 peptide D97. Fresh isolated PBMC from healthyvolunteers were induced in vitro with HER-2 peptides. T-cell surfacephenotypes were determined after one (1) and two (2) stimulations withthe same peptide. Immunofluorescence analysis was performed as describedin the Materials and methods. Symbols indicate (O-O) CD3⁺ cells, (▪-▪)CD8⁺ cells, and (□-□) CD4⁺ cells.

[0058]FIG. 7B. Surface phenotype of T-cells from PBMC culturesstimulated with HER-2 peptide D121. Studies were performed as describedin the legend to FIG. 7A.

[0059]FIG. 7C. Surface phenotype of T-cells from PBMC culturesstimulated with HER-2 peptide C85. Studies were performed as describedin the legend to FIG. 7A.

[0060]FIG. 8A. CTL induction by HER-2 D97 peptide. PBMC from donor 20were induced in vitro with mock stimulated medium only (20.C.2). Aftertwo cycles of stimulation CTL activity was determined in a 4 h ⁵¹Crrelease assay using as targets C1R:A2 cells pulse-labelled with theindicated peptides (D97, D9, D99) or in the absence of peptide (none).

[0061]FIG. 8B. CTL induction by HER-2 D97 peptide. PBMC from donor 20were induced in vitro with D97 (20.D97.2), at a ratio of 3:1. Studieswere performed as described in the legend to FIG. 8A.

[0062]FIG. 8C. CTL induction by HER-2 D97 peptide. PBMC from donor 20were induced in vitro with D97 (20.D97.2), at a ratio of 6:1. Studieswere performed as described in the legend to FIG. 8A.

[0063]FIG. 9A. CTL induction by HER-2 peptides D96 and D97. PBMC fromdonor 20 were stimulated two times with D96 (20.D96.2). CTL activity wasdetermined in a 4 h ⁵¹Cr release assay using as targets C1R:A2 cellswithout addition of exogenous peptide (control) or pulse-labelled withD96 (□), D97 (▪), or NK sensitive targets K562 cells were used as anadditional control.

[0064]FIG. 9B. CTL induction by HER-2 control peptide D95 (▴) peptides.NK sensitive targets K562 cells were used as an additional control.

[0065]FIG. 9C. CTL induction by HER-2 peptides. PBMC from donor 30 wereinduced with D97 peptide (30.D97.1). Seven days later CTL activity ofthese cells was determined using as targets the peptide used forstimulation (D97) or two HLA-A2 binding peptides with unrelated sequenceD113 and D119, as specificity controls.

[0066]FIG. 10A. CTL induction by HER-2 peptide D113. PBMC from threehealthy donors (20, 25 and 30) were induced with D113 peptide. Eachculture was restimulated with D113 once. One week later CTL activity wasdetermined using as targets C1R:A2 cells pulsed with D113. The effectorsare designated as 20.113.2, 25.113.2 and 30.113.2 to indicate the donornumber, the peptide symbol and the number of stimulations with peptide.Experimental conditions were as described in Example 2 and the legendsto FIG. 8A and FIG. 9A. E:T ratios were 20:1 (heavy stripes) and 10:1(medium stripes).

[0067]FIG. 10B. CTL induction by HER-2 peptide D113. PBMC from threehealthy donors (20, 25 and 30) were induced with D113 peptide. Eachculture was restimulated with D113 once. One week later CTL activity wasdetermined using as targets C1R:A2 cells pulsed with control D119peptide. The effectors are designated as 20.113.2, 25.113.2 and 30.113.2to indicate the donor number, the peptide symbol and the number ofstimulations with peptide. Experimental conditions were as described inExample 2 and the legends to FIG. 8A and FIG. 9A. E:T ratios were 20:1(heavy stripes) and 10:1 (medium stripes).

[0068]FIG. 11A. CTL induction by HER-2 peptides D121 and D119. PBMC froma healthy donor (20) were induced with D121:HER-2:392-410 orD119:HER-2:402-410 by stimulating with the peptides twice (20.121.2) oronce (25.121.1 and 25.119.1 respectively). CTL activity was determinedusing C1R:A2 targets pulsed either with the Ag of interest (D119) orcontrol peptides D95, D99, D97, C85 (Table 5). E:T ratios were 20:1(heavy stripes), 10:1 (medium stripes) and 3:1 (light stripes).

[0069]FIG. 11B. CTL induction by HER-2 peptides D121 and D119. PBMC froma healthy donor (25) were induced with D121:HER-2:392-410 orD119:HER-2:402-410 by stimulating with the peptides once (25.121.1). CTLactivity was determined using C1R:A2 targets pulsed either with the Agof interest (D119) or control peptides D95, D99, D97, C85 (Table 5). E:Tratios were 20:1 (heavy stripes), 10:1 (medium stripes) and 3:1 (lightstripes).

[0070]FIG. 11C. CTL induction by HER-2 peptides D121 and D119. PBMC froma healthy donor (25) were induced with D121:HER-2:392-410 orD119:HER-2:402-410 by stimulating with the peptides once (25.119.1). CTLactivity was determined using C1R:A2 targets pulsed either with the Agof interest (D119) or control peptides D95, D99, D97, C85 (Table 5). E:Tratios were 20:1 (heavy stripes), 10:1 (medium stripes) and 3:1 (lightstripes).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] 1. Molecular Therapies for Cancer

[0072] Development of molecular therapies for cancer have historicallyfocused on specific recognition of Ags by cellular immune effectors. Thepresent invention discloses novel strategies aimed at identification ofpeptide targets for CTLs, and generation of T-cell immunity againstspecific epitopes (for a review of T-cell specific immunity, see, e.g.,Ioannides et al., 1992; Houbiers et al., 1993).

[0073] To achieve this, the present invention provides novel naturally-and synthetically-derived peptides which bind human leucocyte antigen-(HLA) class I heavy chains. Appropriate criteria for epitope selectionin vitro have been defined. Using HER-2 protein (which has been proposedas a candidate for an anti-tumor immune response in breast and ovariancancer) these novel peptides have been identified, isolated away fromintact HER-2 protein and characterized. Additionally, synthetic peptidesbased on immunogenic epitopes of the HER-2 protein have also beenproduced.

[0074] Although the dominant anchors for peptide binding to HLA-A2 areLeu (P2) and Val (P9), a number of residues with similar charge and sidechains such as Ile and Met were identified in CTL epitopes from viralproteins (Falk et al., 1991; Bednarek et al., 1991). Analysis of theHER-2 polypeptide sequence identified a large number of nonapeptidesmeeting these criteria (Table 1). With few exceptions, all HLA-A2binding peptides identified in the present invention contain Rothbard'sepitope-motifs. In a few instances, however, the peptide sequencecontained between HLA-A2 anchors matched or overlapped with amphiphilicareas.

[0075] Using more stringent selection criteria, in which only Leu/Ilewere accepted at amino acid position 2 of the peptide (AA₂) and at leastone additional anchor was required, seventeen novel sequences werefound, 10 of which contained Leu and Val at AA₂ and AA₉ respectively.Most of these sequences (shown in Table 2) were adjacent to potentialamphiphilic sites.

[0076] Because it is well-known that not all HLA-A2 anchor-containingpeptides are antigenic, and that it was generally considered notpossible to generate antigens from very short peptide sequences (suchas, e.g., peptides shorter than eight amino acids) the discovery by theinventors that these nonameric peptides both recognized CTLs, stimulatedthem, and produced an immune response was indeed a surprising discovery.

[0077] Three criteria of epitope selection and identified the effects ofpeptide length and presence of anchors on reactivity of HLA-A2 withMA2.1 mAb. MA2.1 mAb recognizes an epitope made of residues 62-65 of theα1 helix which is left to the center of the binding site on HLA-A2(Santon-Aguado et al., 1988). Therefore exogenous peptide binding toHLA-A2 may have three potential consequences:

[0078] (a) induction of a conformational epitope by binding to an‘empty’ HLA-A2 molecule, or displacing a pre-existing endogenous peptidein which case MA2.1 mAb reactivity with HLA-A2 will increase;

[0079] (b) prevention of reactivity of MA2.1 with its epitope either byobscuring residues with which the mAb may interact or interfering withmAb epitope interaction, in which case MA2.1 mAb reactivity with itsepitope will decrease (Hogquist et al., 1993); and,

[0080] (c) no effect in reactivity of MA2.1 mAb with HLA-A2 in whichcase the exogenous added peptide may displace the existing endogenouspeptide, but the conformation of the ‘face’ made of α-peptide-α2 willnot change. In this case conformational changes on the MHC heavy chainmay be detected in a different position using another mAb such as BB7.2which interacts with an epitope containing W(108) (Salter et al., 1987).

[0081] Another surprising aspect of the invention was the fact that whenlong peptides (such as, e.g., peptides longer than 20 amino acids) wereused which contained within their sequences peptide sequences which aredisclosed herein, these >20 amino acid peptides failed to induce changesin FL1 while the novel compositions disclosed herein, effectivelyinduced FL1 changes. This suggests that peptides >20 amino acids (1)either fail to bind to MHC heavy chain because of low affinity, (2) failto be processed to shorter peptides because of either absence ofextracellular proteases secreted by T2 cells or (3) lack the correctsites in the substrate for processing by extracellular proteases.

[0082] The highest increase in FL1 was induced by a D113 analogcontaining G (P1) replacing the bulky and hydrophilic H (P1), suggestingthat residues at P1 may interfere either with mAb or peptide binding.Val (P9) appeared to be important for MA2.1 epitope induction becausesubstitution M→V (P9) induced an increase in FL1 compared with thewild-type nonapeptide C85 (971-979).

[0083] The D97 reactive CTLs identified, as well as the previouslydemonstrated C85 reactive CTLs, indicate that T-cells reactive withthese epitopes are not clonally deleted, while the possible anergicstate of self-reactive CTLs from peripheral blood may have been overcomeby using PBMC at high density as APC. The use of PBMC as APC may haveselective advantages over T2 or C1R:A2 used in other studies (Fisk etal., 1994; Houbiers et al., 1993). First, a number of cells from PBMCcan either present Ag, or release lymphokines, or in general providehelp for CTL induction; second, they reflect closer the situationencountered during in vivo vaccination with tumor peptides thanT2/C1R:A2 cells; and third, induction of peptide reactive T-cell may notonly identify epitopes able to induce a response to a tumor Ag but alsore-stimulate in vivo primed T-cells. These cells can either recognize,or cross-react with epitopes from HER-2 or from other proteins whichmimic the corresponding HER-2 epitopes; fourth, by determining thefrequency of such responses among healthy HLA-A2⁺ donors, this may allowidentification of changes in the responder frequency in breast andovarian cancer patients with HER-2 high and HER-2 low expression ontheir tumors.

[0084] No direct correlation could be demonstrated between the abilityof these peptides to affect the MA2.1 epitopes and either their abilityto stimulate lymphocyte proliferation or to induce in vitro CTLsspecific for the peptide of interest. Both D113 and D119 as well aslonger peptides when used as immunogen to stimulate PBMC in vitro failedto induce a sustained Ag specific CTL response. In cytotoxicity assays,the PBMC cultures stimulated with these peptides failed to showpreferential recognition of Ag used for stimulation. In Example 1 it isshown that HER-2:968-981 or HER-2:971-979 peptides can induce a CTLresponse in vitro. Therefore the inability of peptides HER-2:2:48-56 andHER-2:402-410 to induce in vitro Ag specific CTL may reflect: (1) clonaldeletion of epitope reactive CD8⁺ CTL; (2) anergy or suppression ofspecific CTL clones; or (3) inefficient Ag presentation in the sensethat the peptide although increases the number of MA2.1 epitopes andapparently stabilizes HLA-A2 its conformation does not provide efficientsignaling through TCR (Hogquist et al., 1993).

[0085] 2. CTL Epitopes

[0086] CTL epitopes reported to date are mainly derived from foreign(viral) proteins with little or no homology with self-proteins. Withrespect to CTL responses to self-proteins, it is expected that T-cellsexpressing TCR with high affinity for self-peptide-MHC class I complexesare eliminated in the thymus during development. Self-peptides elutedfrom HLA-A2.1 molecules of various cell lines show residues at P3-P5 andP7-P8 which are different from the sequences of viral epitopesrecognized by human CTLs. Since these residues are likely to contact andinteract with TCR, they may reflect peptides for which autologousT-cells are already tolerant/anergic.

[0087] For T-cell recognizing self-epitopes to be eliminated oranergized, a precondition exists that the peptide-MHC complex is stableenough to engage a sufficient number of TCRs, or at least more stablethan other HLA-A2 peptide complexes, where one peptide can be easilydisplaced by other peptides. Consequently this would suggest that forself-proteins with extension to HER-2, the ones that can bind TCR withhigh affinity during development will be less likely to be recognizedlater when expressed on a tumor other target, than peptides that bindHLA-A2 with low affinity, which under appropriate conditions (e.g., highprotein concentration) may occupy a higher number of HLA-A2 molecules.For low-affinity peptides, modification of the anchors resulting instabilization of peptide—HLA-A2 interaction by replacing weak withdominant anchor residues (e.g., (P9) M→V, should facilitate thereactivity of CTL with targets expressing such antigens, because TCRinteracts mainly with the sequence P4-P8.

[0088] Tumor progression and metastasis are often associated withoverexpression of specific cellular proteins. Epitopes of non-mutatedoverexpressed proteins can be targets of a specific cellular immuneresponse against tumor mediated by T-cells. Moreover, when T-cellepitopes are present, distinction between tumor immunity/autoimmunityand unresponsiveness can be predicated on the protein concentration as alimiting factor of epitope supply. The present inventors havedemonstrated that CTLs from patients with ovarian tumors whichover-express HER-2 proto-oncogene can recognize both autologous tumorand novel synthetic analogs of a specific HER-2 epitopes. These epitopeswere identified in HER-2 containing nonapeptides with HLA-A2 anchors.Analysis of potential amphiphilic sites identified natural peptides andnovel synthetic peptides which surprisingly affected the reactivity ofconformationally-dependent HLA-A2 specific monoclonal antibodies (mAbs),and indicated specific binding of these peptides similar to that seenfor HER-2 epitopes.

[0089] 3. Screening Kits

[0090] In another aspect, the present invention contemplates adiagnostic kit for screening samples suspected of containing HER-2/neuor neu-related polypeptides, or cells producing such polypeptides. Saidkit can contain a peptide or antibody of the present invention. The kitcan contain reagents for detecting an interaction between an agent and apeptide or antibody of the present invention. The provided reagent canbe radio-, fluorescently- or enzymatically-labeled. The kit can containa known radiolabeled agent capable of binding or interacting with apeptide or antibody of the present invention.

[0091] In another aspect, the present invention contemplates adiagnostic kit for detecting CTLs. The kit comprises reagents capable ofdetecting a peptide of the present invention and a CTL. The providedreagent may also be radio-, enzymatically-, or fluorescently-labeled.The kit can contain a radiolabeled peptide capable of binding to orinteracting with a CTL, or may contain a radiolabeled antibody capableof binding to or interacting with a peptide of the present inventionwhich in turn interacts with a CTL. The kit can contain a polynucleotideprobe from about 15 to 60 nucleotides that encodes a peptide of thepresent invention or any of their complements. The kit can contain anantibody immunoreactive with a peptide of the present invention.

[0092] The reagent of the kit can be provided as a liquid solution,attached to a solid support or as a dried powder. Preferably, when thereagent is provided in a liquid solution, the liquid solution is anaqueous solution. Preferably, when the reagent provided is attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent provided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

[0093] 4. Immunodetection Kits

[0094] In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that theCTL-stimulating peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect CTLs or neu-related epitope-containing peptides. Ingeneral, these methods will include first obtaining a sample suspectedof containing such a protein, peptide or antibody, contacting the samplewith an antibody or peptide in accordance with the present invention, asthe case may be, under conditions effective to allow the formation of animmunocomplex, and then detecting the presence of the immunocomplex.

[0095] In general, the detection of immunocomplex formation is quitewell known in the art and may be achieved through the application ofnumerous approaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

[0096] For diagnostic purposes, it is proposed that virtually any samplesuspected of comprising either the HER-2/neu peptide or neu-relatedpeptides or antibody sought to be detected, as the case may be, may beemployed. Exemplary samples include clinical samples obtained from apatient such as blood or serum samples, ear swabs, sputum samples,middle ear fluid or even perhaps urine samples may be employed.Furthermore, it is contemplated that such embodiments may haveapplication to non-clinical samples, such as in the titering of antigenor antibody samples, in the selection of hybridomas, and the like.

[0097] In related embodiments, the present invention contemplates thepreparation of kits that may be employed to detect the presence ofHER-2/neu or neu-related proteins or peptides and/or antibodies in asample. Generally speaking, kits in accordance with the presentinvention will include a suitable CTL-stimulating peptide or an antibodydirected against such a protein or peptide, together with animmunodetection reagent and a means for containing the antibody orantigen and reagent. The immunodetection reagent will typically comprisea label associated with the antibody or antigen, or associated with asecondary binding ligand. Exemplary ligands might include a secondaryantibody directed against the first antibody or antigen or a biotin oravidin (or streptavidin) ligand having an associated label. Of course,as noted above, a number of exemplary labels are known in the art andall such labels may be employed in connection with the presentinvention.

[0098] The container means will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably aliquotted. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, andreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

[0099] 5. ELISAs

[0100] ELISAs may be used in conjunction with the invention. In an ELISAassay, proteins or peptides incorporating dgA antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

[0101] After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of about25° to about 27° C. Following incubation, the antisera-contacted surfaceis washed so as to remove non-immunocomplexed material. A preferredwashing procedure includes washing with a solution such as PBS/Tween®,or borate buffer.

[0102] Following formation of specific immunocomplexes between the testsample and the bound antigen, and subsequent washing, the occurrence andeven amount of immunocomplex formation may be determined by subjectingsame to a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween®).

[0103] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonicacid [ABTS] and H₂O₂, in the case of peroxidase as the enzyme label.Quantification is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

[0104] 6. Epitopic Core Sequences

[0105] The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-HER-2/neuantibodies.

[0106] As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-HER-2/neuantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a HER-2 proto-oncogene polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the HER-2 polypeptide will alsobind to, react with, or otherwise recognize, the cross-reactive peptideor protein antigen. Various immunoassay methods may be employed inconjunction with such antibodies, such as, for example, Westernblotting, ELISA, RIA, and the like, all of which are known to those ofskill in the art.

[0107] The identification of CTL-stimulating immunodominant epitopes,and/or their functional equivalents, suitable for use in vaccines is arelatively straightforward matter. For example, one may employ themethods of Hopp, as taught in U.S. Pat. No. 15 4,554,101, incorporatedherein by reference, which teaches the identification and preparation ofepitopes from amino acid sequences on the basis of hydrophilicity. Themethods described in several other papers, and software programs basedthereon, can also be used to identify epitopic core sequences (see, forexample, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No.4,554,101). The amino acid sequence of these “epitopic core sequences”may then be readily incorporated into peptides, either through theapplication of peptide synthesis or recombinant technology.

[0108] Preferred peptides for use in accordance with the presentinvention will generally be on the order of 8 to 20 amino acids inlength, and more preferably about 8 to about 15 amino acids in length.It is proposed that shorter antigenic CTL-stimulating peptides willprovide advantages in certain circumstances, for example, in thepreparation of vaccines or in immunologic detection assays. Exemplaryadvantages include the ease of preparation and purification, therelatively low cost and improved reproducibility of production, andadvantageous biodistribution.

[0109] It is proposed that particular advantages of the presentinvention may be realized through the preparation of synthetic peptideswhich include modified and/or extended epitopic/immunogenic coresequences which result in a “universal” epitopic peptide directed toHER-2/neu and neu-related sequences. These epitopic core sequences areidentified herein in particular aspects as hydrophilic regions of theHER-2/neu proto-oncogene polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

[0110] An epitopic core sequence, as used herein, is a relatively shortstretch of amino acids that is “complementary” to, and therefore willbind, antigen binding sites on transferrin-binding protein antibodies.Additionally or alternatively, an epitopic core sequence is one thatwill elicit antibodies that are cross-reactive with antibodies directedagainst the peptide compositions of the present invention. It will beunderstood that in the context of the present disclosure, the term“complementary” refers to amino acids or peptides that exhibit anattractive force towards each other. Thus, certain epitope coresequences of the present invention may be operationally defined in termsof their ability to compete with or perhaps displace the binding of thedesired protein antigen with the corresponding protein-directedantisera.

[0111] In general, the size of the polypeptide antigen is not believedto be particularly crucial, so long as it is at least large enough tocarry the identified core sequence or sequences. The smallest usefulcore sequence anticipated by the present disclosure would generally beon the order of about 8 amino acids in length, with sequences on theorder of 9 or 10 being more preferred. Thus, this size will generallycorrespond to the smallest peptide antigens prepared in accordance withthe invention. However, the size of the antigen may be larger wheredesired, so long as it contains a basic epitopic core sequence.

[0112] The identification of epitopic core sequences is known to thoseof skill in the art, for example, as described in U.S. Pat. No.4,554,101, incorporated herein by reference, which teaches theidentification and preparation of epitopes from amino acid sequences onthe basis of hydrophilicity. Moreover, numerous computer programs areavailable for use in predicting antigenic portions of proteins (seee.g., Jameson & Wolf, 1988; Wolf et al., 1988). Computerized peptidesequence analysis programs (e.g., DNAStar Software, DNAStar, Inc.,Madison, Wis.) may also be useful in designing synthetic peptides inaccordance with the present disclosure.

[0113] Syntheses of epitopic sequences, or peptides which include anantigenic epitope within their sequence, are readily achieved usingconventional synthetic techniques such as the solid phase method (e.g.,through the use of commercially available peptide synthesizer such as anApplied Biosystems Model 430A Peptide Synthesizer). Peptide antigenssynthesized in this manner may then be aliquotted in predeterminedamounts and stored in conventional manners, such as in aqueous solutionsor, even more preferably, in a powder or lyophilized state pending use.

[0114] In general, due to the relative stability of peptides, they maybe readily stored in aqueous solutions for fairly long periods of timeif desired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4° C., or more preferably, frozen. Of course, where the peptides arestored in a lyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

[0115] 7. Immunoprecipitation

[0116] The antibodies of the present invention are particularly usefulfor the isolation of antigens by immunoprecipitation.Immunoprecipitation involves the separation of the target antigencomponent from a complex mixture, and is used to discriminate or isolateminute amounts of protein. For the isolation of membrane proteins cellsmust be solubilized into detergent micelles. Nonionic salts arepreferred, since other agents such as bile salts, precipitate at acid pHor in the presence of bivalent cations.

[0117] In an alternative embodiment the antibodies of the presentinvention are useful for the close juxtaposition of two antigens. Thisis particularly useful for increasing the localized concentration ofantigens, e.g. enzyme-substrate pairs.

[0118] 8. Western Blots

[0119] The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

[0120] Immunologically-based detection methods for use in conjunctionwith Western blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

[0121] 9. Vaccines

[0122] The present invention contemplates vaccines for use in bothactive and passive immunization embodiments. Immunogenic compositions,proposed to be suitable for use as a vaccine, may be prepared mostreadily directly from immunogenic CTL-stimulating peptides prepared in amanner disclosed herein. Preferably the antigenic material isextensively dialyzed to remove undesired small molecular weightmolecules and/or lyophilized for more ready formulation into a desiredvehicle.

[0123] The preparation of vaccines which contain peptide sequences asactive ingredients is generally well understood in the art, asexemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all incorporated herein byreference. Typically, such vaccines are prepared as injectables. Eitheras liquid solutions or suspensions: solid forms suitable for solutionin, or suspension in, liquid prior to injection may also be prepared.The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccines.

[0124] Vaccines may be conventionally administered parenterally, byinjection, for example, either subcutaneously or intramuscularly.Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides: suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10%, preferably about 1to about 2%. Oral formulations include such normally employed excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 10 to about 95% of active ingredient,preferably about 25 to about 70%.

[0125] The peptides of the present invention may be formulated into thevaccine as neutral or salt forms. Pharmaceutically-acceptable salts,include the acid addition salts (formed with the free amino groups ofthe peptide) and those which are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0126] The vaccines are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, e.g., the capacity of theindividual's immune system to synthesize antibodies, and the degree ofprotection desired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. However,suitable dosage ranges are of the order of several hundred microgramsactive ingredient per vaccination. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

[0127] The manner of application may be varied widely. Any of theconventional methods for administration of a vaccine are applicable.These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection or the like. The dosage of thevaccine will depend on the route of administration and will varyaccording to the size of the host.

[0128] Various methods of achieving adjuvant effect for the vaccineincludes use of agents such as aluminum hydroxide or phosphate (alum),commonly used as about 0.05 to about 0.1% solution in phosphate bufferedsaline, admixture with synthetic polymers of sugars (Carbopol®) used asan about 0.25% solution, aggregation of the protein in the vaccine byheat treatment with temperatures ranging between about 70° to about 101°C. for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with a 20% solution of a perfluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

[0129] In many instances, it will be desirable to have multipleadministrations of the vaccine, usually not exceeding six vaccinations,more usually not exceeding four vaccinations and preferably one or more,usually at least about three vaccinations. The vaccinations willnormally be at from two to twelve week intervals, more usually fromthree to five week intervals. Periodic boosters at intervals of 1-5years, usually three years, will be desirable to maintain protectivelevels of the antibodies. The course of the immunization may be followedby assays for antibodies for the supernatant antigens. The assays may beperformed by labeling with conventional labels, such as radionuclides,enzymes, fluorescents, and the like. These techniques are well known andmay be found in a wide variety of patents, such as U.S. Pat. Nos.3,791,932; 4,174,384 and 3,949,064, as illustrative of these types ofassays.

[0130] 10. DNA Segments Encoding Novel Peptides

[0131] The present invention also concerns DNA segments, that can beisolated from virtually any mammalian source, that are free from totalgenomic DNA and that encode the novel peptides disclosed herein. DNAsegments encoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like ofHER-2/neu-related or other non-related gene products. In addition theseDNA segments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

[0132] As used herein, the term “DNA segment” refers to a DNA moleculethat has been isolated free of total genomic DNA of a particularspecies. Therefore, a DNA segment encoding a CTL-stimulating peptiderefers to a DNA segment that contains CTL-simulation coding sequencesyet is isolated away from, or purified free from, total genomic DNA ofthe species from which the DNA segment is obtained. Included within theterm “DNA segment”, are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, viruses, and the like.

[0133] Similarly, a DNA segment comprising an isolated or purifiedCTL-stimulating peptide-encoding gene refers to a DNA segment which mayinclude in addition to peptide encoding sequences, certain otherelements such as, regulatory sequences, isolated substantially away fromother naturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, polypeptide- or peptide-encoding unit. As will be understoodby those in the art, this functional term includes both genomicsequences, cDNA sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides orpeptides.

[0134] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case, a gene encoding CTL-stimulatingpeptides, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or cDNA coding regions. Of course, this refers tothe DNA segment as originally isolated, and does not exclude genes orcoding regions later added to the segment by the hand of man.

[0135] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences that encodea CTL-stimulating peptide species that includes within its amino acidsequence an amino acid sequence essentially as set forth in any of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.

[0136] The term “a sequence essentially as set forth in any of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29”means that the sequence substantially corresponds to a portion of thesequence of either SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, or SEQ ID NO: 29 and has relatively few amino acids that are notidentical to, or a biologically functional equivalent of, the aminoacids of any of these sequences. The term “biologically functionalequivalent” is well understood in the art and is further defined indetail herein (for example, see Preferred Embodiments). Accordingly,sequences that have between about 70% and about 80%, or more preferablybetween about 81% and about 90%, or even more preferably between about91% and about 99% amino acid sequence identity or functional equivalenceto the amino acids of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, and SEQ ID NO: 29 will be sequences that are “essentiallyas set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, and SEQ ID NO: 29.”

[0137] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding regionor may include various internal sequences, i.e., introns, which areknown to occur within genes.

[0138] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding either of the peptidesequences disclosed in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, and SEQ ID NO: 29, or that are identical to orcomplementary to DNA sequences which encode any of the peptidesdisclosed in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQID NO: 29, and particularly those DNA segments disclosed in SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ IDNO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64. For example,DNA sequences such as about 14 nucleotides, and that are up to about1,000, about 500, about 200, about 100, about 50, and about 25 basepairs in length (including all intermediate lengths) are alsocontemplated to be useful.

[0139] It will be readily understood that “intermediate lengths”, inthese contexts, means any length between the quoted ranges, such as 14,15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

[0140] It will also be understood that this invention is not limited tothe particular nucleic acid sequences which encode peptides of thepresent invention, or which encode the amino acid sequences of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29,including those DNA sequences which are particularly disclosed in SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64.Recombinant vectors and isolated DNA segments may therefore variouslyinclude the peptide-coding regions themselves, coding regions bearingselected alterations or modifications in the basic coding region, orthey may encode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

[0141] The DNA segments of the present invention encompassbiologically-functional equivalent peptides. Such sequences may arise asa consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

[0142] If desired, one may also prepare fusion proteins and peptides,e.g., where the peptide-coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins that may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0143] Recombinant vectors form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment, whether encodinga full length protein or smaller peptide, is positioned under thecontrol of a promoter. The promoter may be in the form of the promoterthat is naturally associated with a gene encoding peptides of thepresent invention, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

[0144] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a DNA segment encoding aCTL-stimulating peptide in its natural environment. Such promoters mayinclude promoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, eukaryotic, or mammalian cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriate promotersystems contemplated for use in high-level expression include, but arenot limited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

[0145] In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of CTL-stimulatingpeptides or epitopic core regions, such as may be used to generateanti-peptide antibodies, also falls within the scope of the invention.DNA segments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful.

[0146] In addition to their use in directing the expression ofCTL-stimulating peptides of the present invention, the nucleic acidsequences contemplated herein also have a variety of other uses. Forexample, they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment any of SEQID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64 willfind particular utility. Longer contiguous identical or complementarysequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000(including all intermediate lengths) and even up to full lengthsequences will also be of use in certain embodiments.

[0147] The ability of such nucleic acid probes to specifically hybridizeto peptide-encoding sequences will enable them to be of use in detectingthe presence of complementary sequences in a given sample. However,other uses are envisioned, including the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

[0148] Nucleic acid molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so, identical or complementary to DNA sequencesof any of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQID NO: 64, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. Smaller fragments willgenerally find use in hybridization embodiments, wherein the length ofthe contiguous complementary region may be varied, such as between about10-14 and about 100 nucleotides, but larger contiguous complementaritystretches may be used, according to the length complementary sequencesone wishes to detect.

[0149] The use of a hybridization probe of about 10-14 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 10 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

[0150] Of course, fragments may also be obtained by other techniquessuch as, e.g., by mechanical shearing or by restriction enzymedigestion. Small nucleic acid segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.Nos. 4,683,195 and 4,683,202 (each incorporated herein by reference), byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

[0151] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of 50° C. to 70° C. Such selective conditionstolerate little, if any, mismatch between the probe and the template ortarget strand, and would be particularly suitable for isolatingCTL-stimulating peptide-encoding DNA segments. Detection of DNA segmentsvia hybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1993; Segal 1976; Proskop, 1991; and Kuby, 1991, are particularlyrelevant.

[0152] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate CTL-stimulatingpeptide-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0153] In certain embodiments, it will be advantageous to employ nucleicacid sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase, instead of radioactive or other environmental undesirablereagents. In the case of enzyme tags, colorimetric indicator substratesare known that can be employed to provide a means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

[0154] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridization as well as in embodiments employing a solid phase. Inembodiments involving a solid phase, the test DNA (or RNA) is adsorbedor otherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

[0155] 11. Biological Functional Equivalents

[0156] Modification and changes may be made in the structure of thepeptides of the present invention and DNA segments which encode them andstill obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thefollowing codon table: TABLE 1 Amino Acids Codons Alanine Ala A GCA GCCGCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acidGlu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0157] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventors that various changes may be made in the peptide sequences ofthe disclosed compositions, or corresponding DNA sequences which encodesaid peptides without appreciable loss of their biological utility oractivity.

[0158] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0159] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0160] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0161] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

[0162] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0163] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

[0164] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0165] 12. Site-Specific Mutagenesis

[0166] Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the DNA. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 17 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues on both sides of the junctionof the sequence being altered.

[0167] In general, the technique of site-specific mutagenesis is wellknown in the art, as exemplified by various publications. As will beappreciated, the technique typically employs a phage vector which existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage are readily commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis whicheliminates the step of transferring the gene of interest from a plasmidto a phage.

[0168] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex isformed,wherein one strand encodes the original non-mutated sequence andthe second strand bears the desired mutation. This heteroduplex vectoris then used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0169] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis isprovided as a means of producing potentially useful species and is notmeant to be limiting as there are other ways in which sequence variantsof peptides and the DNA sequences encoding them may be obtained. Forexample, recombinant vectors encoding the desired peptide sequence maybe treated with mutagenic agents, such as hydroxylamine, to obtainsequence variants.

[0170] 13. Monoclonal Antibody Generation

[0171] Means for preparing and characterizing antibodies are well knownin the art (See, e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; incorporated herein by reference).

[0172] The methods for generating monoclonal antibodies (mAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

[0173] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0174] As is also well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0175] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster, injection mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs.

[0176] mAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified LTBP-3 protein, polypeptide orpeptide. The immunizing composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep frogcells is also possible. The use of rats may provide certain advantages(Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

[0177] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are aselected for use in the mAb generating protocol. These cells may beobtained from biopsied spleens, tonsils or lymph nodes, or from aperipheral blood sample. Spleen cells and peripheral blood cells arepreferred, the former because they are a rich source ofantibody-producing cells that are in the dividing plasmablast stage, andthe latter because peripheral blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

[0178] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0179] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

[0180] One preferred murine myeloma cell is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

[0181] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 ratio, though the ratio may vary from about20:1 to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described by Kohler andMilstein (1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding pp. 71-74, 1986).

[0182] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0183] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B-cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B-cells.

[0184] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immuno-bindingassays, and the like.

[0185] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

[0186] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Oligopetide Induction of a Cytotoxic T Lymphocyte Response toHER-2/neu Proto-oncogene in Vitro

[0187] A. Materials and Methods

[0188] 1. Peptides

[0189] HER-2 peptides were prepared by the Synthetic Antigen Laboratoryof M.D. Anderson Cancer Center (Houston, Tex.) using Merrifield'ssolid-phase system and a peptide synthesizer (Ioannides et al., 1993).All reagents were of high purity (>99%) and obtained from MilliporeCorporation. Eluted peptides were transferred in aqueous solution bypassing over Sephadex G-25 columns and lyophilized. Crude syntheticpeptides were separated by reverse-phase HPLC. Identity and purity ofthe final materials were established by amino acid analysis.Purification yielded single peaks by analytical HPLC and the purity ofpeptides used in these studies was ≧97%.

[0190] 2. Immunofluorescence

[0191] mAbs to CD3 (OKT3-FITC), CD4 (OKT4-FITC), and CD8 (OKT8-FITC)were obtained from Ortho Diagnostic (Ortho, Raitan, N.J.); mAb W6/32(anti-HLA, -A, -B, -C) was from Dako (Dako-Dakopatts, Denmark); and mAbLeu1 1a (anti CD16) was obtained from Beckton-Dickinson (Mountain View,Calif.). mAb BB7.2 and MA2.1 (anti-HLA-A2)-producing clones were fromATCC, mAb Ab2 against HER-2/neu was obtained from Oncogene Science(Manhasset, N.Y.). Immunofluorescence studies were performed asdescribed (Ioannides et al., 1993).

[0192] 3. Cells and Cell Lines

[0193] Tumor lines and leukocytes of the donors of ovarian malignantascites were phenotyped for HLA-A, B, and C antigens by the blood bankat M.D. Anderson Cancer Center, Leukocytes of PBMC donors used asresponder cells (HLA-A, B, C) were typed at the HistocompatibilityLaboratory of the Methodist Hospital (Houston, Tex.). The HLA types ofthe donors are presented in Table 2. Expression of HLA-A2 on ovariantumors, fibroblasts, and EBV-B cell lines (HLA-A2 transfectants) wasconfirmed by immunofluorescence using culture supernatant from mAb MA2.1(Ioannides et al., 1993).

[0194] C1R:A2, C1R:A1, and C1R:A3 cells express transfected genomicclones of HLA-A2.1, HLA-A1, and HLA-A3. These cells were obtained fromDr. William E. Biddison, National Institute of Neurological Disorders,Bethesda, Md. C1R (Class I reduced) is a mutant cell line that does notexpress HLA-A2 (Bednarek et al., 1991; Gammon et al., 1992). These cellswere maintained in complete RPMI 1640 medium containing 100 μg/mlL-glutamine, 40 μg/ml gentamicin, and 10% fetal calf serum (FCS)(RPMI-FCS). Ovarian tumors and lines of known HLA phenotype used inthese studies were: SKOV3 (HLA-A3, 28, B18, 35, Cw5), OVA-1 (HLA-A1, 24,B8, 35, Cw4), OVA-14 (HLA-A2, 30, B14, 44, Cw2.8), OVA-16 (A2, 19 B8,35), OVA-24 (HLA-A2, 24, B8, 51, Cw2, 7), and (VA-31 (HLA-A11, -, B60,62, Cw3). Additional targets used in this study were the EBV-B cell lineXxCr (HLA-A2, -, B7, 8, Cw7) and the breast carcinoma line SKBr3(HLA-A11, -, B18, 40, Bw22). SKBr3 overexpressing HER-2 was obtainedfrom Dr. Mien Chie-Hung, Department of Tumor Biology, M.D. AndersonCancer Center. TABLE 2 HLA TYPING OF LYMPHOCYTE DONORS HLA type No.Donor number A B C 1. 30  2, 33 14, 35 w4 2. 41  1, 2  8, — w7 3. 51  1,2  8, — w7 4. 46  2, 2 18, 60 w3 5. 86  2, 2 18, 61 w3 6. 14 32, — 41,51 N.D.^(a) 7. 15  1, 32  8, 35 w4, w7

[0195] Ovarian tumors were separated from TIL/TAL by centrifugation overFicoll-Hypaque gradients, as previously described (Ioannides et al.,1991), and stored frozen in aliquots in liquid nitrogen until used.Ovarian tumor lines were maintained in culture in L-15 medium (Gibco,Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 20μg/ml gentamycin. Ovarian CTL-TAL lines autologous with OVA-1, OVA-14,OVA-16, and OVA-31 have been generated as described from lymphocytesinfiltrating malignant ascites (TAL) by coculture of tumors with TAL inRPMI-FCS in the presence of 25-50 U/ml of IL-2 (Cetus, Emeryville,Calif.) and 250 U/ml of tumor necrosis factor-α (TNF-α) (Genentech, SanFrancisco, Calif.) (Ioannides et al., 1991).

[0196] 4. Transfection of Ovarian Tumor Line SKOV3 with HLA-A2

[0197] The HLA-A2 expression vector RSV.5-neo containing HLA-A2.1full-length cDNA was provided by Drs. Richard V. Turner and William E.Biddison. The RSV.5-neo expression vector is a derivative of RSV.3(Jacobson et al., 1989). The SKOV3 cell line was cloned by stringentlimiting dilution (Ioannides et al., 1993), and individual clones weretransfected with the plasmid using the Lipofectin reagent and procedure(Gibco-BRL, Gaithersburg, Md.) as described by the manufacturer.Transfectants were selected in culture with 800 μg/ml of G418 (SigmaChemical Co., St. Louis, Mo.). Surface expression of HLA-A2 wasdetermined by immunofluorescence with MA 2.1 mAb as described (Ioannideset al., 1993). Several clones that expressed high levels of HLA-A2 suchas 2B6 (SKVO3.A2) were selected for cytotoxicity studies.

[0198] 5. Cytotoxicity Assays

[0199] Tumor cells and fibroblasts were labeled with 200 μCi of ⁵¹Cr(Na⁵¹CrO₄; Amersham, Arlington Heights, Ill.) for 90 min at 37° C.(Ioannides et al., 1991). Lymphoblastoid cells and HLA-A2 transfectantswere labeled overnight in RPMI-FCS, then washed three times andincubated with effector cells in RPMI-FCS in an incubator with 5% CO₂(Bednarek et al., 1991; Gammon et al., 1992). When peptide recognitionwas determined, targets were incubated with 25 μM of peptides overnightduring ⁵¹Cr labeling or with 10 μM peptide for 2 h at 37° C. in RPMI-FCSthen washed three times before being incubated with effector cells.Separate controls for spontaneous and total lysis were made for eachpeptide-pulsed target (Ioannides et al., 1991; Bednarek et al., 1991;Gammon et al., 1992). After 4-5 h, 100 μl of supernatant was collectedand counted. To determine maximum lysis in 20-h assays, plates were leftundisturbed in the incubator and the supernatant was collected afterovernight incubation. For cold target inhibition studies, C1R:A2 cellswere preincubated with HER-2 or control peptides overnight, then washedand admixed with ⁵¹Cr-labeled targets at 2:1 and 6:1 (cold:hot targets)ratios. Percentage lysis was calculated from the formula:100×[(E−S)/(T−S)], where E is experimental release, S is release in theabsence of CTL, and T is release in 2 M HCl.

[0200] 6. Generation of in Vitro HER-2 Peptide-reactive CTL

[0201] CTL cultures reacting with HER-2 peptides were generatedfollowing procedures described for in vitro induction of influenzamatrix and tum- peptide-specific CTL (Bednarek et al., 1991; Gammon etal., 1992; Alexander et al., 1991) with several modifications. In brief,PBMC from HLA-A2⁺ and HLA-A2⁻ donors were separated by Ficoll-Hypaque™gradient centrifugation. PBMC (5-10×10⁶) were washed, resuspended in afinal volume of 100-250 μl in PBS, and incubated with the stimulatingpeptide for 90 min at 37° C. The final concentration of the stimulatingpeptide ranged between 5 and 50×10⁻⁶ M. Afterwards, cells wereirradiated (4000 rad), washed, and plated in wells of 24-well plates(Costar, Cambridge, Mass.) in 2.0 ml at a final concentration of0.5-1.0×10⁶ cells/ml. As responding cells, autologous PBMC were added ata final concentration of 1.0-1.5×10⁶/ml. Sequences of HER-2 peptideanalogs used for stimulation or specificity determination are presentedin Table 3.

[0202] Cultures were initiated in RPMI 1640 medium containing 100 μg/mlL-glutamine, 40 μg/ml gentamycin, and 5% heat-inactivated andsterile-filtered human AB plasma (RPMI-HS). After 3 days, 5 U of IL-2(Cetus) was added in each well. One unit of IL-2 (Cetus) equals 6 IU ofIL-2 (Ioannides et al., 1991). After 2 additional days one-third of themedium from each well was replaced with an equal volume of RPMI-HScontaining 15 U/ml of IL-2. Four days later, cells were removed fromcultures, washed, and restimulated either with irradiated freshautologous PBMC or C1R:A2 cells pulsed with HER-2 additional days theexpanding cultures were restimulated with peptides following theprocedures described above. Five to 6 days after the second stimulationand 7 to 8 days after the third and subsequent stimulations, cultureswere tested for cytotoxic activity against C1R:A2 cells pre-pulsed withHER-2 peptides and unrelated control peptides containing HLA-A2 anchormotifs.

[0203] Cultures that showed higher lysis of targets pulsed with HER-2peptides than control peptides were maintained for further studies.These cultures were propagated and expanded by periodic cycles ofrestimulation with peptide-pulsed fresh autologous PBMC asantigen-presenting cells. After the fourth stimulation cells weregradually adapted to growth in RPMI-FCS by replacing 25% of the culturemedium every 3 days with RPMI-FCS over a period of 2 weeks. CD3⁺CD8⁺CD4⁻cells were isolated from bulk CTL cultures by positive selection onanti-CD8 mAb-coated culture flasks (AIS Micro CELLector, Applied ImmuneSciences, Menlo Park, Calif.) as described (Letessier et al., 1991).Isolated CD8⁺ cells were restimulated with HER-2 peptide-pulsed PBMC,either autologous or in some instances allogeneic that matched onlyHLA-A2 with the responding cells. TABLE 3 SEQUENCES OF PEPTIDES SequencePeptide^(a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SEQ ID NO: 1. C43 R F R E LV S E F S R M A R 65 2. C85 E L V S E F S R M 7 3. C84 E L V S E F S R V6 4. C44 R F R E L I I E F S R M A R 66 5. D132 Muc-1:16-1 S +TL,14 L A+TL,18 D P A H G V 67 6. D125 Muc-1:8-17 G L T S A P D T R V 68

[0204] B. Results

[0205] 1. Generation of in Vitro HER-2 Peptide Reacting CTLs

[0206] To define conditions for in vitro CTL induction by stimulationwith HER-2 peptides of PBMC from healthy volunteers, two syntheticpeptides were used for priming: (1) C43 (HER-2:968-981)=RFRELVSEFSRMAR(SEQ ID NO: 31), which contains as HLA-A2 anchors Leu(972) at P2 and Met(979) at P9, includes two Rothbard epitope motifs ELVS and RMAR and mostof the amphiphilic area 968-984; and (2) C84(HER-2:971-979(Val)=ELVSEFSRV (SEQ ID NO: 6) where Met(P9) has beensubstituted by Val because Val is the dominant anchor residue at P9 andalthough it does not contact the TCR (Malden et al., 1993), itstabilizes the HLA-A2-HER-2 peptide complex. Leu and Met were also foundin CTL epitopes at P9, as indicated by sequence information (Parker etal., 1992). These peptides were selected because of our previousobservations that tumor reacting CTL-TAL isolated from lymphocytesinfiltrating ovarian malignant ascites can recognize synthetic peptidesderived from the highly amphiphilic area HER-2:968-984 on HLA-A2⁺targets (Ioannides et al., 1993). All cultures were initiated in RPMI-HSto avoid induction of T-cells reactive with determinants on FCSproteins. In contrast, cytotoxicity assays were performed in RPMI-FCS tominimize interferences from recognition by CTL of human proteins (Wolfelet al., 1993).

[0207] The ability of PBMC cultures to recognize peptides used forpriming was determined by measuring the lysis of peptide-pulsed C1R:A2cells. Three out of five individual cultures tested lysed C1R:A2 targetspulsed with C43, C84, or both. Results with two representative donors(No. 41 and No. 51) are shown in FIG. 1. It should be mentioned thatthese two donors were siblings and had identical HLA phenotype. A commonfeature of C43- and C84-induced cultures was that they showed minimallysis of C1R:A2 cells in 4-h cytotoxicity assays but significantdifferences were observed between lysis of peptide pulsed and controlC1R:A2 targets in 20-h assays at 2-3 weeks after stimulation. Whencytotoxicity was determined early (1 week after stimulation), in certaininstances, they showed high background lysis.

[0208] Interestingly, all cultures stimulated with the C84 peptideshowed similar levels of lysis of either C43- or C84-pulsed targets in20-h cytotoxicity assays (FIG. 1A, FIG. 1B, FIG. 1C, FIG. 2A, and FIG.2B).

[0209] HER-2 peptide-stimulated PBMC cultures tend to lose specificityover time and that the numbers of CD8⁺ cells tend to decrease, due toovergrowth of CD4⁺ cells. CD8⁺ cells were isolated from bulk CTLcultures from donor 41 by positive selection on anti-CD8 mAb-coatedplates. The resulting cells were 100% CD3⁺, 97% CD8⁺, and 1% CD4⁺.Separated CD8⁺ cells were propagated in culture by repeated stimulationswith C43 and C84 peptide-pulsed PBMC and expanded in medium containing15-25 U/ml of IL-2 for more than 6 months. The 41.CD8⁺ CTL linerecognized both C43 and C84 peptides and at a much lesser extent,control D125 and D132 peptides. These peptides contain HLA-A2 anchorsintroduced by us but differ in sequence form HER-2 peptides (Table 3).The absence of HLA-A2 anchors in the natural sequence of D125 and D132suggests that they are not presented to corresponding CTL in humans. Thesequences of D125 and D132 were chosen from Muc-1 core sequence (Gendleret al., 1988). When C43 and C84 peptides were preincubated with C1R:A1cells (HLA-A1 transfectants which expressed only HLA-A1), 41.CD8⁺ CTLfailed to elicit a higher lysis of peptide pulsed than of controltargets.

[0210] Similar results were observed with HLA-A3 transfectants (FIG. 2Aand FIG. 2B). These results suggested that 41.CD8⁺ CTL line is peptideAg specific. Similar results were obtained with PBMC from donor 30stimulated with C43/C84 peptides. HLA-A2⁺ transfectants did notcross-present C43/C85/C84 to HLA-A2⁻ CTL from donors 14 and 15 (Table 3)induced with the same peptides. This may suggest that recognition ofC43/C84 is HLA-A2 restricted. C43/C85/C84 lack anchor residues forHLA-A1:E(P3), P(P4) and Y(P9); HLA-B8:K(P3), R(P5) and L(P9) (Dibrino etal., 1994), as well as for HLA-B35:Pro(P2) and Tyr(P9) (Hill et al.,1992).

[0211] 2. HER-2 Peptide-Induced CD8⁺ Cells can Lyse Ovarian TumorsOverexpressing HER-2 Proto-oncogene

[0212] In vitro peptide-induced CTL cultures can recognize HER-2peptides used as immunogen. The major question with respect to thespecificity of in vitro-induced CTL is whether they canspecifically-lyse targets endogenously expressing the antigen ofinterest. To address this question, the ability of HER-2peptide-stimulated CTL to lyse ovarian tumors overexpressing HER-2protein was investigated. The ability of 41.CD8⁺ CTL line to lyse anovarian tumor (OVA-16) overexpressing HER-2 was tested usingNK-sensitive targets as lysability controls. OVA-16 tumor shared HLA-A2with donor 41 effectors (Table 2). The ability of 41.CD8⁺ effectors tolyse OVA-16 was determined at 4 and 20 h. The results are shown in FIG.3A and FIG. 3B. As expected from the results presented in FIG. 1A, FIG.1B, FIG. 1C, FIG. 2A and FIG. 2B, lysis of OVA-16 by 41.CD8⁺ effectorsin 4-h assays was low, although higher than K562. In 20-h cytotoxicityassays, lysis of OVA-16 was significantly higher than K562 cells.

[0213] To determine whether susceptibility of ovarian tumors to lysiscorrelates with levels of HER-2 protein expression on tumor, the abilityof the 41.CD8⁺ CTL line to lyse two HLA-2⁺ fresh isolated ovarian tumorsOVA-16 and OVA-14 was tested. The results are presented in FIG. 3C. Bothshared only HLA-A2 with the donor 41, but they differed at the levels ofexpression of HER-2. Immunofluorescence staining with theanti-HER-2-specific mAb showed 77.5% HER-2⁺ cells with a meanfluorescence intensity (MFI) of 28.67 for OVA-16 and 17.4% HER-2⁺ cellswith a MFI of 6.2 for OVA-14. They were designated as HER-2^(high) andHER-2^(low) respectively. The control HLA-A target, ovarian tumor lineSKOV3 (99% HER-2⁺) was also designated as HER-2^(high).

[0214] The 41.CD8⁺ line showed significantly higher lysis ofHER-2^(high) than HER-2^(low) targets, suggesting that lysis of HER-2expressing ovarian tumors may be dependent on Ag density. Lysis of theOVA-14 tumor was similar to that of control XxCr and C1R:A2 cells(HLA-A2⁺, HER-2⁻) and SKBr3 (HLA-A2⁻, HER-2^(high)). The SKOV3 tumor(HLA-A2⁻, HLA-A3⁺, HER-2^(high)) was lysed at levels comparable withcontrol lines, suggesting that HER-2 recognition requires presentationby HLA-A2, because SKOV3.A2 targets were recognized. Both OVA-14 andOVA-16 expressed comparable levels of HLA-A2 antigens on the surface asdetermined by immunofluorescence with MA2.1 mAb (92.1% HLA-A2 positivecells and 49.0 mean fluorescence for OVA-14 and 85.3% HLA-A2 positivecells and 42.0 mean fluorescence for OVA-16, respectively). In separatestudies, OVA-16, OVA-14, and SKOV3 tumors were efficiently lysed by LAKcells, suggesting that there were no major differences in theirlysability by cytolytic effectors.

[0215] It is unlikely that tumor killing by 41.CD8⁺ CTL reflects LAKtype activity. LAK cells lyse K562 with higher efficiency than they lysehuman tumors (Grimm et al., 1982). Also, both C1R:A2 and SKOV3.A2 weretransfected with the same HLA-A2 plasmid expression vector. Therefore,lysis of SKOV3.A2 but not of C1R:A2 suggests that 41.CD8⁺ CTL are notonly HLA-A2 reactive but also Ag reactive. In separate studies, LAKcells lysed effectively both C1R:A2 and T2 cells. This lysis was notaffected by C43/C84 or mutated peptides based on this sequence. Theseresults show that HER-2 peptide-induced CD8⁺ cells from human PBMC canrecognize targets endogenously expressing HER-2 protein.

[0216] To confirm that the 41.CD8⁺ CTL line recognizes epitopes onHER-2^(high) tumors contained on peptides used for stimulation, coldtarget inhibition studies were performed. In an attempt to inhibit lysisof the OVA-16 tumor by 41.CD8⁺ CTL with either C43- or C85-(thewild-type HER-2 peptide 971-979) pulsed C1R:A2 cells were used withC1R:A2 cells alone or pulsed with the D125 peptide as controls. Theresults are shown in FIG. 4A and FIG. 4B. Inhibition of OVA-16 lysis bythe 41.CD8⁺ line was observed in both 4- and 20-h assays. C43- andC85-pulsed C1R:A2 cells but not specificity controls, C1R:A2 cells aloneor pulsed with the D125 peptide,inhibited the lysis of the OVA-16 tumor.As expected, levels of lysis were lower in 4-h versus 2-h assays.Increasing the cold:hot ratio to 6:1 did not significantly increase theinhibitory effects of the HER-2 peptide-pulsed C1R:A2. That highlyspecific, but incomplete, inhibition was observed here and in otherhuman CTL systems (Jerome et al., 1993) reflect low Ag (peptide) densityon targets use for inhibition or an increase in background nonspecificlysis as observed in FIG. 3C.

[0217] These peptides were recognized by autologous tumor reactiveCTL-TAl, suggesting the presence on the tumor of similar orcross-reactive CTL epitopes (Ioannides et al., 1993). To address whetherthese peptides interfere with tumor lysis by autologous tumor reactiveCTL-TAL in HLA-A2⁻ systems, the lysis of OVA-1, HER-2^(high) (HLA-A1,24, B8, 35, Cw4) and OVA-31, HER-2^(high) was determined by pre-pulsingwith either C43 or as a control, C44 peptide (VS→II). Target lysis byCTL-1 was: OVA-1 (68%), K562 (18%), OVA-1 plus C43 (37%), and OVA-1 plusC44 (51%). C43 significantly inhibited by 45% lysis of OVA-1 by CTL-1,while less inhibition (25%) was observed with C44. However, C43 and C44had no effect on lysis of OVA-31 by CTL-31. This suggested that thesepeptides can bind certain MHC Class I heavy chains other than HLA-A2 andcan interfere with lysis of certain HLA-A2⁻ tumors by autologous CTLs.

[0218] Therefore CD8⁺ CTL lines can be induced in vitro with HER-2peptide analogs and lyse ovarian tumors overexpressing HER-2. It is alsolikely that a T-cell epitope with a sequence similar or cross-reactingwith peptide analogs from the area HER-2:968-981 is associated withHLA-A2 on the tumor cell surface.

[0219] C. Discussion

[0220] Evidence has been presented showing that human PBMC from 10healthy volunteers can be primed in vitro with HER-2 peptide analogs todevelop lymphocyte cultures with Ag-specific CTL activity. A CD8⁺ CTLline developed from bulk cultures recognized not only peptides used asimmunogen but also ovarian tumors endogenously expressing HER-2.Peptide-induced CD8⁺ CTL lysed targets endogenously expressing HER-2 butnot K562 cells, an ovarian tumor expressing low levels of HER-2.Furthermore, based on the ability of C1R:A2 cells pulsed with C43, C85,or C84 to inhibit HER-2^(high) tumor lysis compared with the inabilityof C1R:A2 cells alone or pulsed with D125 to mediate the sameinhibition, the findings demonstrate that HER-2 peptide-induced CD8⁺ CTLrecognizes similar or cross-reactive epitopes on tumors expressingHER-2. At similar levels of HLA-A2 expression efficiency of tumor lysiswas dependent on the levels of HER-2 expression.

[0221] The weak lysis observed in 4-hr assays does not reflect “slow”lysis. Slow lysis rarely achieves target lysis above 500% at E:T ratiosof 60:1 in 20- to 24- hr assays (Ratner and Clark, 1993). CTL showedlevels of lysis in the range of 60-80% at 10:1 or even 5:1 E:T ratios.One possibility to be considered is that the frequency of HER-2 reactiveclones in peptide-induced CTLs is relatively low and they diluted amongnon-cytotoxic cells. The 41.DD8⁺ line secreted TNF-α when coculturedovernight with C1R:A2 cells in the presence, but not in the absence, ofHER-2 peptides. TNF-α secretion was inhibited by HLA-A2 specific MA2.1mAb, suggesting that peptide recognition associated with HLA-A2 isneeded for lymphokine secretion. With respect to the efficiency of thesepeptides for target sensitization for maximum lysis this was observedwhen targets were preincubated with 5 μM peptide for 1 hr or culturedwith 25 μM peptide overnight. The amount of peptide bound on HLA-A2⁺molecules cannot be estimated, however, by comparing with other reportson human CTL assays performed in the presence of peptide in solution,these CTLs needed 2-3×10²-fold more peptide for similar levels of targetrecognition, but in 20-hr assays (Gammon et al., 1992; Schmidt et al.,1991; Kos and Müllbacher, 1992; Stauss et al., 1992; Anderson et al.,1992). This peptide concentration is significantly less than the10⁷-fold difference in peptide concentration needed for efficient Agrecognition reported for murine CTL induced in vivo and in vitro bypeptides (Schild et al., 1991).

[0222] It may be possible that if HLA-A2 acts as a restriction elementfor specific HER-2 peptides, TCR with high affinity for these naturalpeptides may be eliminated during thymic selection, leaving only TCRwith low affinity (Bowness et al., 1993). The only conservativesubstitution introduced to strengthen the P9 anchor (Met→Val) had noinhibitory effects in peptide-stimulating ability or CTL specificity.TCR contacts mainly residue in the sequence P4-P7, while P2 and P9 areburied in the HLA-A2 binding pockets (Madden et al., 1993). Of interest,the 14mer peptide C43 had similar sensitizing ability for lysis oftargets as the shorter peptide C84. Although it may be possible thatactivity in C43 is associated with the presence of contaminatingpeptides at levels lower than the ability of detection, several otherpossibilities need to be taken in consideration: proteolytic degradationas extracellular processing occurs and the longer peptides are bettersubstrates than shorter than peptides for proteolysis (Sherman et al.,1992). This may also suggest a role for the group RFR and/or thecarboxy-terminal R in Ag processing before HLA-A2 binding.

[0223] Since targets were always pulsed with the same concentrations ofpeptides, the kinetics of target recognition may also reflect differenteffects of factors involved in in vitro priming of T-cells with Ag. Ithas been previously shown that by increasing both responder cell and Ag(peptide) density, murine Ag-specific CTL can be induced in vitro. TheseCTL recognized targets which endogenously expressed the Ag of interest(Winter et al., 1991).

[0224] The experience with in vitro induction of human CTLs by peptideis limited. Recent reports have shown that Ag-specific CTLs can beinduced in vitro using peptide analogs of EBV nuclear antigens (EBNA)(Schmidt et al., 1992), influenza matrix (Bednarek et al., 1991; Gammonet al., 1992), or Plasmodium falciparum pre-erythrocytic stage antigens(Hill et al., 1992). Given the frequency of EBV and influenza infectionsit is possible that they represented, at least in some instances,secondary CTL responses of in vivo-primed T-cells. Based on molecularmimicry between self and foreign proteins at the three and tetrapeptidelevels (Ohno, 1991), it is not unlikely that naturally processed T-cellepitopes from self-proteins may be cross-reactive (Anderson et al.,1992).

[0225] Since HER-2 is a self-antigen, HER-2 reactive T-cells may beprimed in vivo and non-deletional mechanisms of tolerance in theperiphery may render HER-2-primed T-cells anergic or suppressed.However, a recent report demonstrated that Ag-reactive T-cellstransferred in Ag-reactive T-cells transferred in Ag tolerant transgenicmice can be recovered, suggesting that tolerance induction in theperiphery may not affect primed T-cells and that the lack ofauto-reactivity may be because of the low levels of antigen expressed onnormal cells (Hu et al., 1993). The HER-2 proto-oncogene product isexpressed at low levels in normal cells of origin. Results suggest thatin vivo priming to HER-2 epitopes is possible when HER-2 is expressed at100- to 200-fold higher than normal levels (Ioannides et al., 1993). Incontrast with viral infections which essentially turn off the hostprotein synthesis to favor the expression of virally coded polypeptides,overexpression of HER-2 does not generally inhibit the tumor's proteinsynthesis. Thus, additional antigens are expected to compete with HER-2for HLA-A2 binding and presentation to TCR.

[0226] PBMC from 5 of 11 healthy HLA-A2⁺ volunteers tested showed CTLresponses to HER-2 peptides used for priming, and CTLs and tumor cloneshave been developed to identify HER-2 epitopes recognized by tumorreactive CTLs.

EXAMPLE 2 Sequence Motifs of Human HER-2 Proto-Oncongene Important forPeptide Binding to HLA-A2

[0227] A. Materials and Methods

[0228] 1. Peptides

[0229] HER-2 peptides were synthesized as described in Example 1. Thepurity of peptides used in these studies was ≧97.

[0230] 2. Immunofluorescence

[0231] mAbs to CD3 (OKT3-FITC), CD4 (OKT4-FITC) and CD8 (OKT8-FITC) wereobtained from Ortho Diagnostic (Ortho, Raritan, N.J.), mAb W6/32(anti-HLA, -A, -B, -C) from Dako (Dako-Dakopatts, Denmark); HLA-A2reacting mAb BB7.2 and MA2.1 from ATCC. Immunofluorescence studies wereperformed as described in Example 1.

[0232] 3. HLA-typing

[0233] Leukocytes of the PBMC donors used as responder cells were typedby the Blood Bank at M.D. Anderson Cancer Center. The HLA-types were asfollows: donor 20: HLA-A 2, 11, B35, 51, Cw7, donor 25: HLA-A 2, 3, B44,60; donor 30: HLA-A2, 33 B14, 35, Cw4. Expression of HLA-A2 on HLA-A2transfectants was confirmed by immunofluorescence using culturesupernatant from mAb MA2.1 (Ioannides et al., 1993).

[0234] 4. Cytotoxicity Assays

[0235] Target cells were labeled with ⁵¹Cr (Na⁵¹CrO₄; Amersham,Arlington Heights, Ill.) for 90 min at 37° C. (Ioannides et al., 1991;Ioannides et al., 1991), or overnight in RPMI medium, containing 10%FCS, 100 82 g/ml L-glutamine and 40 μg/ml gentamycin (RPMI-FCS), thenwashed and incubated with the effector cells in complete RPMI-FCS in anincubator with 5% CO₂. Targets were incubated either with 25 μM ofpeptide overnight during labeling, or with 10 μM peptide for 2 h at 37°C. in RPMI-FCS, then washed three times before being incubated witheffector cells. Separate controls for spontaneous and total lysis oftargets were made for each peptide pulsed target (Ioannides et al.,1993; Fisk et al., 1994; Gammon et al., 1992). After 4-5 h of incubation100 μl of supernatant were collected and counted. Percent lysis wascalculated from the formula: 100×[(E−S)/(T−S)], were E=experimentalrelease, S=release in the absence of CTL, T=release in 2 M HCl.

[0236] 5. Target Cells and Cell Lines

[0237] The human lymphoblastoid cell lines C1R and T2 have beenpreviously described (Gammon et al., 1992; Bednarek et al., 1991; Salterand Creswell, 1986; Anderson et al., 1993). C1R (Class I reduced) is amutant cell line that does not express HLA-A2, C1R:A2 cells expresstransfected genomic clones of HLA-A2.1. These cells were obtained fromDr. William E. Biddison (National Institute of Neurological Disorders,Bethesda, Md.). T2 (transport deletion mutant) cells were obtained fromDr. Peter Creswell (Yale University School of Medicine, New Haven,Conn.). C1R:A2 cells were maintained in RPMI-FCS. T2 cells weremaintained in Iscove's Modified Dulbecco Medium (IMDM) containing 5%Fetal Calf Serum (IMDM-FCS).

[0238] 6. Generation of in vitro HER-2 Peptide-reactive CTL

[0239] CTL cultures were generated following the procedures describedfor in vitro induction of influenza matrix and tum peptide specific CTL(Gammon et al., 1992; Bednarek et al., 1991; Salter and Creswell, 1986)with several modifications. In brief, PBMC from HLA-A2⁺ donors wereseparated by Ficoll-Hypaque™ gradient centrifugation. 5-10'10⁶ PBMC wereresuspended in a final volume of 100-250 μl in PBS and incubated withthe stimulating peptide at a final concentration between 5-50×10⁻⁶ M for90 min at 37° C. Afterwards, cells were irradiated (4000 rad), washed,and plated in wells of 24 well plates (Costar, Cambridge, Mass.) in 2.0ml at a final concentration of 0.5-1.0×10⁶ cells/ml. As responders,autologous PBMC were added at a final concentration of 1.0-1.5×10⁶/ml.

[0240] Cultures were initiated in RPMI 1640 medium containing 100 μg/mlL-glutamine, 40 μg/ml gentamycin and 5% heat-inactivated and sterilefiltered human AB plasma (RPMI-HS). The use of human serum duringstimulation and culture and of FCS during CTL assays was intended toavoid induction of FCS peptide reactive CTLs. After three days, 5 U ofIL-2 (Cetus) equals 6 IU of IL-2 (Ioannides et al., 1991). Two dayslater, one third of the medium was replaced with an equal volume ofRPMI-HS containing 15 U/ml of IL-2. Four days later, cells wererestimulated with irradiated fresh autologous PBMC pulsed with the samepeptides. Three days later, 5 U of IL-2 (Cetus) was added to each well.The expanding cultures were subjected to a second round of restimulationas described above. Six days after the first and second stimulations andseven to eight days after the third stimulation, cultures were testedfor cytotoxic activity against C1R:A2 cells pulsed with eitherstimulating peptides or unrelated control peptides. Control cultureswere established without HER-2 peptides, containing the same number ofautologous stimulators and responders PBMC.

[0241] 7. Proliferation Assays

[0242] Fresh PBMC from healthy volunteers isolated by Ficoll-Hypaque™were distributed into 96-well round-bottomed plates (Falcon,Becton-Dickinson) at 2×10⁵/well in RPMI-FCS. Peptides were added at 50μg/ml. The studies were performed at least twice using PBMC from thesame donor, in quadruplicate. After 5 days, for the last 16 h inculture, wells were pulsed with 1 μCi of [³H]-thymidine (³H-Tdr) andcounted. Proliferation was determined as ³H-Tdr incorporation and c.p.m.determined in the samples of PBMC cultured with peptides and weredivided by c.p.m. determined same cultures in the absence of peptides,to determine the stimulation index (S.I.).

[0243] B. Results

[0244] 1. Selection of Candidate Antigenic Peptides from HER-2 PeptidesPredicted by Algorithmic Methods

[0245] Sequence analysis for the presence of potentially amphiphilicareas revealed a small number (<20) of potential sites capable offorming long amphiphilic α-helices over 3-4 turns (12 residues) in the1255 residue sequence of HER-2 (Ioannides et al., 1993). A number ofshorter sequences have also been identified. Most of these sequencescontained Rothbard's epitope motifs (Rothbard and Taylor, 1988). Sincethe focus was peptides presented by HLA-A2, these regions as well as theentire HER-2 sequence were searched for areas containing the predicted,as well as the alternatively reported, HLA-A2 anchors: i.e. L/M/I/V (P2)and V/L/M/I (P9) (Parmiani, 1993; Bednarek et al., 1991; Parker et al.,1992). Several areas were found to meet all three criteria of selection.These areas are as follows:

[0246] (a) HER-2: 968-984, which not only forms a perfect amphiphilichelix but also contains two Rothbard's epitope motifs and a nonapeptidewith predicted HLA-A2 anchors. This area has been previously found to berecognized by tumor reactive CTL (Ioannides et al., 1993).

[0247] (b) The area HER-2:41-56 contains L(43), L(49) and the groupVV(55-56). This corresponds to two overlapping potentially HLA-A2binding peptides: an octapeptide HER-2:42-49 followed by a nonapeptideHER-2:48-56. The presence of HL and VV groups renders these peptideshighly hydrophobic, and consequently they have low solubility in PBS orculture medium. With respect to the sequence HER-2:48-56, thecorresponding synthetic peptide (D113) had low solubility in PBS. DMSOup to 50% was used for rapid solubilization. The analogsD114=HER-2:47-56(48H→L) and D115=HER-2:48-56(48H→G) were designed in anattempt to improve solubility and increase the ability of exogenouslysupplied peptides to bind to HLA-A2. To overcome these problems at leastin part, peptides D96=HER-2:4-54 and D97=HER-2:42-51 were synthesized,which, although longer than the minimum HLA-A2 binding peptide, arewater-soluble.

[0248] (c) The area HER-2:391-411 contains two potentially HLA-A2binding nonapeptides: HER-2:391-399 (PLQPEQLQV) (SEQ ID NO: 12) andHER-2:402-410 (TLEEITGYL) (SEQ ID NO: 13). An octapeptide HER-2:396-403with HLA-A2 anchors at P2 and P8:QLQVFETL (SEQ ID NO: 14) is nested inthe sequence and overlaps with the carboxy- and amino terminal regionsof the HER-2:391-399 and 402-410.

[0249] Two other areas containing decapeptides: HER-2:344-353=GLGMEHLREV(SEQ ID NO: 15) and HER-2:1089-1098=DLGMGAAKGL (SEQ ID NO: 16) bothinclude predicted HLS-A2 anchors but not overlapping or continuousepitopes. Several other areas also show potential amphiphilic sites andinclude Rothbard's epitope motifs. While these areas do not includeHLA-A2 anchor motifs, they may, however, include anchors for otherHLA-types.

[0250] 2. Peptides Identified by the Presence of HLA-A2 Anchors

[0251] In addition to the sites identified by the overlap of potentiallyamphiphilic sites and Rothbard's epitope motifs, a number of peptidescan be identified in the sequence of HER-2 by the presence of HLS-A2anchors at positions 2 and 9. A large number of sites (>35) containingnonapeptides with: dominant, strong or weak P2 and P9 anchors predictedor reported for HLA-A2 (Falk et al., 1991) were found in the HER-2sequence. The sequences of most of these peptides are presented in Table4. Additional nonapeptides are found in the Leu and Val richtransmembrane domain (655-675). In addition to nonamers, a large numberof octa- and decamers were found in the HER-2 sequence containingL/I/V/M as HLA-A2 anchors. These sequences are not included in Table 4except in a few cases where octa- and decamers are part of epitopeclusters. In addition to clustered potential HLA-A2 binding peptidesfrom the signal (Ioannides et al., 1992; Ioannides et al., 1993;Parmiani, 1993; Slamon et al., 1989; Fisk et al., 1994; Falk et al.,1991; Rothbard and Taylor, 1988; DeLisi and Berzofsky, 1985; Stauss etal., 1992) and transmembrane (655-675) areas, putative HLA-A2 bindingpeptides are clustered either as continuous or overlapping peptides asfollows: 42-91 (two 8- and three 9-mers), 141-179 (three 9-mers),391-419 (three 9-mers), continued with 423-474 (six 9-mers), 781-807(three 9-mers and one 10-mer), 828-859 (one 8-mer and four 9-mers).

[0252] In certain areas, the last two carboxyterminal residues of aputative HLA-A2 binding peptide overlap with the first two aminoterminalresidues of the next peptide because P2 and P9 anchors are the same orsimilar (L/V). Most of the peptides include Rothbard's epitope motifs.However most of the nonapeptides either do not derive from longamphiphilic areas, or are highly hydrophobic according to theirsequence; when their sequence is viewed on axial projection (Edmundson'swheel) (Kaiser and Kezdy, 1984) the majority of the peptides (28/38)show limited segregation of hydrophilic and hydrophobic residues.

[0253] Crystallographic analysis of the LSA-A2 peptide complex revealsan additional binding pocket in HLA-A2 accommodating a hydrophobicresidue in position 6 and the likelihood that residues in positions 4and 8 are hydrophilic and oriented upwards (towards TDR) (Saper et al.,1991; Madden et al., 1993). None of the nonapeptides of sequences shownin the Table 4 contains all the additional strong anchors in thepositions 4, 6, and 8 identified by Rammensee and collaborators (Falk etal., 1991). However at least 3/17 nonamers contain one additional strongHLA-A2 anchor and 11/18 nonamers contains at least two additional weakHLA-A2 anchors (Table 5). For peptide selection the following groupswere considered equivalent: L and I at P2, R and K at Pa, P4, P5 and P8,L and M at P9, either because of TABLE 4 HER-2/NEU PEPTIDES CONTAININGP2 AND P9 HLA-A2 ANCHORS^(a) From Amino Acid To Amino Acid Peptide No.Position # Position #  1  5  13  2  42  49  3  48  56  4  76  84  5  84 91  6 141 149  7 160 168  8 171 179  9 369 377 10 391 399 11 402 410 12411 419 13 423 431 14 435 443 15 442 450 16 447 455 17 457 465 18 466474 19 596 604 20 603 611 21 627 635 22 650 658 23 689 697 24 747 755 25781 790 26 789 797 27 793 801 28 799 801 29 828 836 30 835 842 31 838846 32 845 853 33 851 859 34 883 891 35 904 912 36 971 979 37 986 994 381172  1180 

[0254] structural similarities, or because they have been reported to bepart of CTL epitopes (Parker et al., 1992).

[0255] Sequence analysis of HLA-A2 bound peptides shows an alternationof hydrophobic and hydrophilic residues. P2 and P3 are generally made ofhydrophobic residues, P4 of hydrophilic residues, while the charge andhydropathy of residues in P5-P9 alternate, following in general thepattern: P5 (variable/neutral)—P6 (hydrophobic)—P7 (variable)—P8(hydrophilic)—P9 (always hydrophobic) (Falk et al., 1991). Although thepeptide is bound to HLA in an extended conformation stabilized withhydrogen bonds, the alternation between hydrophobic and hydrophilicresidues is in general agreement with Rothbard's epitope motifs and withhypotheses that certain T-cell epitopes are derived from amphiphilicsites.

[0256] Examination of the physicochemical properties of HER-2 peptidesmay assist in predicting which of the peptides shown in Table 4 willbind HLA-A2. However this would not address whether these self-peptidesare capable of activating T-cells. To gain insight into these questions,specific areas were targeted: 41-56, 392-411, and 968-984. Each areacontains a nonapeptide: 48-56 (D113), 402-410 (D119) and 971-979 (C85).All share Leu as P2 anchor. Peptide 43-56 contains the dominant P9anchor Val, while 402-410 contains Leu and 971-979 contains Met whichare expected to be weak anchors. All share a hydrophilic residue at P4:D97 (Q), D119 (E) and C85 (S). Differences are evident in the residuesin the other positions, where only C85 has a strong P8 anchor (R). Thesequences of peptides from these regions are shown in the Table 4.

[0257] 3. Effects of HER-2 Synthetic Analogs on Conformational Epitopeson HLA-A2

[0258] To determine whether these peptides and longer analogs affectHLA-A2 conformation as an indication of HLA-A2 binding, the effects ofHER-2 peptides on the reactivity of conformationally dependent mAb MA2.1and BB.7.2 were examined with HLA-A2 of T2 cells. The human cell line T2has a defect affecting endogenous peptide loading of MHC class Imolecules. As a consequence, cell surface expression of HLA-A2 is lower(30-40%) than in normal LBL lines transfected with HLA-A2 (e.g., C1R)but the reactivity of MA2.1 and BB.7.2 mAb is increased when certainHLA-A2 binding peptides are added to the culture medium (Anderson etal., 1993). Although most human MHC class I molecules cannot be inducedat the low temperatures used for their murine counterparts because offundamental structural differences between human and mouse class I(Anderson et al., 1993), the fact that they express few endogenous(mainly signal) peptides (Zweerink et al., 1993) increases thesensitivity of detection of peptide-HLA-A2 interaction.

[0259] The results of immunofluorescence studies are presented in FIG.5A, FIG. 5B, FIG. 5C, and FIG. 5D. The nonapeptide D113 induced asignificant increase in FL1. As expected, its analogs D114 and D115increased FL1 even further (FIG. 5A). However since they have shown lowsolubility, the study was repeated with peptides dissolved in DMSO. Thenonapeptide D113 induced a significant decrease in the reactivity ofMA2.1 mAb with HLA-A2. Both D114 and D115 were unable to increasereactivity of MA2.1 with HLA-A2. In contrast, D97 which has identicalP1-2 anchors with D113 but nests an octapeptide, D96 which covers theentire area 41-54 with the exception of the VV group (P8-9) of D113 anddecapeptide D99=DLGMGAAKGL (HER-2:1089-1098) (SEQ ID NO: 5) showed aslight increase in FL1 of T2 cells in comparison with control peptides(D98 and D100) or in the absence of peptide. TABLE 5 HER-2 PEPTIDESCONTAINING ADDITIONAL HLA-A2 ANCHORS TO P2 AND P9 Peptide Sequence No.of Anchors SEQ ID No. Position 1 2 3 4 5 6 7 8 9 Strong Weak NO: 1 48-56H L Y Q G C Q V V 2 2 17 2 76-84 D I Q E V Q G Y V 3 1 20 3 141-149 Q LR S L T E I L 1 4 21 4 171-179 D I F H K N N Q L 1 3 22 5 369-377 K I FG S L A F L 1 6 11 6 391-399 P L Q P E Q L Q V 2 1 12 7 402-410 T L E EI T G Y L 2 3 13 8 411-419 Y I S A W P D S L 1 4 23 9 457-465 S L R E LG S G L 2 3 24 10 650-658 P L T S I I S A V 2 2 25 11 689-697 R L L Q ET E L V 2 2 26 12 747-755 K I P V A I K V L 1 4 27 13 789-797 C L T S TV Q L V 3 0 10 14 828-836 Q I A K G M S Y L 2 3 28 15 851-859 V L V K SP N H V 3 1 8 16 971-979 E L V S E F S R M 2 1 7 17 1172-1180 T L S P GK N G V 2 2 29

[0260] TABLE 6 SEQUENCES OF HER-2 PEPTIDES USED IN THIS STUDY HER-2Peptides: SEQ ID Peptide Position Sequence NO: D97 42-51 H L D M L R H LY Q 30 D96 41-54 T H L D M L R H L Y Q G C Q 31 D113 48-56 H L Y Q G C QV V 17 D114 47-56 R L L Y Q G C Q V V^(a) 18 D115 48-56 G L Y Q G C Q VV 19 D98 N Q E V T A W D G T Q R 32 D119 402-410 T L E E I T G Y L 13D120 397-410 L Q V F E T L E E I T G Y L 33 D121 392-411 L Q P E Q L Q VF E T L E E I T G Y L Y 34 D122 396-406 Q L Q V F E T L E E I 35 D95392-404 L Q P E Q L Q V F E T L E 36 C85 971-979 E L V S E F S R M 7 C86971-981 E L V S E F S R M A R 37 C43 968-981 R F R E L V S E F S R M A R38 C44 968-981 R F R E L I I E F S R M A R 39 B69 972-984 L V S E F S RM A R D P Q 40 C61 968-977 R F R E L V S E F S 41 D169 964-972 E C R P RF R E L ^(b) 42 D170 968-984 R F R E L V S 43 D99 1089-1098 D L G M G AA K G L 16 D100 1086-1098 F D G D L G M G A A K G L 45 FBP Peptides: E3725-33 R I A W A R T E L 46 E38 112-120 N L G P W I Q Q V 47 E39 191-199E I W T H S T K V 48 E40 247-255 S L A L M L L W L 49 E41 245-253 L L SL A L M L L 50

[0261] Of peptides from the area 392-410, the analog D119 correspondingto a nonapeptide with dominant P2 and weak P9 anchor showed asignificant increase in FL1 over control T2 cells preincubated withoutpeptide. Interestingly, aminoterminal elongation of the peptide (D120)and elongation followed by truncation (D122) increased the FL1 onlyslightly over the base level. Similarly, a peptide (D121) containing theentire area failed to significantly affect the FL1, suggesting that itis probably not processed by external proteases to shorter fragments ofcorrect length.

[0262] The model nonapeptide C85 (=HER-2:971-979) from the third areafailed to significantly increase FL1 of T2 cells reacting with MA2.1.C85 contains a dominant P2, a strong P8 and weak P9 anchor. This wasalso true for longer analogs B69 and C43. To address the questionwhether this reflects the weakness of the P9, and P6 anchors analogsC84(M→V), C83(RM→KV) and C81(F→V, RM→KV) were synthesized. Peptide C84induced a significant increase in FL1 that was comparable with D119(FIG. 5C), suggesting that the presence of a strong anchor at P9 in thispeptide is important for induction of a MA2.1 conformational epitope onT2. C83 did not increase further the FL1, suggesting that thesubstitution R→K may not be critical for reactivity of MA2.1 withHLA-A2. Of note, C81 significantly increased the FL1, suggesting thatthe presence of V at P6 is important for induction of MA2.1conformational epitopes. Since the previous data suggest that C85 mayinteract with HLA-A2, the reactivity of BB7.2 mAb with T2 cellspreincubated with the same peptides was examined. The results show thatC85 induces an increase in FL1 of cells stained with BB.7.2 (FIG. 5D).The analogs C84 and C81 induced an even higher increase in FL1 of cellsreacted with BB.7.2.

[0263] To clarify whether (L/I (P2) and V/L (P9) as critical elementsfor induction of MA2.1 conformational epitopes is restricted to HER-2, acontrol study analyzed the effect on FL1 of T2 cells stained with MA2.1of five nonapeptide analogs from the sequence of folate binding protein(FBP) which is also overexpressed in ovarian cancer. The results arepresented in FIG. 6. Of five peptides, one (E37) failed to affect MA2.1epitope expression, three showed a moderate increase similar with C84regardless that either Leu or Val were present P9, and only one showed avery high increase in FL1. This peptide (E38) has a different P2 (L vsI) from E39 which showed only a moderate increase in FL1 and includedI(P6). Two other peptides (E40-E41) containing the groups ALM and MLL atP5-P7 failed to induce an increase in FL1. These results indicate thatin addition to the presence of predicted dominant P2 and P9 anchors,induction of conformational MA2.1 epitopes on HLA-A2 also depends on thepeptide sequence at P3-P8. It is likely that the presence of certainresidues affects the reactivity of MA2.1 mAb with HLA-A2. ThereforeMA2.1 epitope expression alone does not necessarily reflect the affinityof peptide binding to MA2.1.

[0264] 4. Stimulation of Peptide Reactive CTL in Vitro

[0265] HER-2 peptides (Table 6) were tested for their ability tostimulate HLA-A2⁺ PBMC to proliferate in vitro. PBMC from healthy donorswere incubated with HER-2 peptides from the groups: 41-56, 392-410 and968-984 for 5 days. With few exceptions, significant cell proliferationwas not observed in all 4 PBMC samples from individual donors ofdifferent HLA-types including HLA-A2. (S.I. ranged between 0.8-1.5)suggesting that these short peptides were not mitogenic. The exceptionto these observations was Donor 20.S.I. for D95 was 6.4, for D121 was4.3, but for the nonamer D119 was only 1.1. Similarly, the S.I. for thelonger peptide D96 (41-54) was 2.8 but for the shorter peptide D97 wasonly 1.5. These differences were statistically significant.Proliferation of PBMC stimulated with peptides in the presence of IL-2failed to clearly distinguish between peptides that induced lymphocyteproliferation and those that did not, because of the overall increase inthe levels of proliferation of both control and peptide stimulatedsamples.

[0266] To address the question of whether in vitro stimulation of PBMCwith HER-2 peptides followed by culture in the presence of IL-2 leads toT-cell phenotype change, the % CD3, CD4, and CD8 expression on thesurface of HER-2 peptide-stimulated PBMC were determined. The results ofa typical study are shown in FIG. 7A, FIG. 7B and FIG. 7C. Nine daysafter the first stimulation with either peptide D97 (a decamercontaining a nested octapeptide), D121 (a 20-mer containing nested anoctapeptide and a nonapeptide) and C85 (nonapeptide) and expansion inIL-2, all cultures showed a significant increase in CD8⁺ cells and adecrease in CD4⁺ cells was observed associated with overall cellexpansion and growth. This trend continued in all cultures and 10-15days after a third stimulation, with the same peptide in all cultures,CD4⁺ cells constituted the dominant (>80%) cell population.

[0267] 5. Lytic Activity and Specificity of HER-2 Peptide-stimulatedPBMC

[0268] To elucidate the ability of HER-2 peptides to induce CTLs invitro, the ability of HLA-A2⁺ PBMC cultured in the presence of HER-2peptides and IL-2 to recognize peptides used as stimulators wasdetermined. HER-2 peptides with different sequences were used asspecificity controls. C1R:A2 cells were used as targets because theyexpress only HLA-A2. A first group of peptides selected as stimulatorswere from the area HER-2:41-56 as follows: D96 and D97 containing theoctapeptide: 42-49, and D113 and D114 corresponding to the overlappingpeptides 48-56 and 47-56. Stimulation and restimulation with irradiatedautologous PBMC pulsed with peptide showed mixed results. In certaincases, higher peptide recognition was determined, in others lack ofpeptide specificity was observed. In most cultures, after the secondstimulation with HER-2 peptides, CD4⁺ cells became the dominantpopulation, and they expressed either LAK type lytic activity or failedto recognize the Ag used for stimulation.

[0269] The results of a typical study that used as targets three HER-2peptides and as effectors PBMC from donor 20 stimulated either once withD97 or cultured in the same conditions in the absence of HER-2 peptide(as control) are shown in FIG. 8A, FIG. 8B, and FIG. 8C. Controlcultures showed low levels of similar lysis of all targets. In contrast,cultures stimulated with D97 showed at 6:1 E/T ratio somewhat higherlysis of targets pulsed with the peptides used for stimulation, than ofcontrol peptides D98 (no HLA-A2 anchors) and D99 but the backgroundlysis was relatively high. Similarly when PBMC from the same donor werestimulated with D96 which includes the area HER-2:42-51, higher lysis oftargets pulsed with D97 than D96 was observed (FIG. 9A, FIG. 9B, andFIG. 9C). The same cultures showed lower lysis of targets pulsed eitherwith control D95 peptide (not used for stimulation), or control C1R:A2cells, or the NK sensitive targets K562 cells. D97 stimulated PBMC fromthe donor 30 showed higher lysis of targets pulsed with D97 than withthe overlapping 48-56 and control D119 nonapeptides. The results areshown in FIG. 9A, FIG. 9B, and FIG. 9C. In 2/3, donors peptides D96/D97induced in vitro CTLs can preferentially recognize the peptide used asstimulator. This suggests that a potential epitope capable ofstimulating T-cells in vitro is nested in the area 42-51.

[0270] Peptide D113 and its mutated analog D114 induced a CTL responsewhich apparently lacked Ag specificity (FIG. 10A and FIG. 10B). Althoughin 2/3 HLA-A2⁺ donors, at certain E:T ratios peptide induced CTL showedhigher recognition of targets pulsed with D113 than of control D119peptide, the differences were minimal. These peptide-induced CTL weredesignated as non-specific. D113, D114 and D115 showed higher increasein reactivity of MA2.1 mAb with HLA-A2 than D96/D97. However theyinduced less specific CTL than D96/D97. The reasons for thesedifferences are unknown, however the results should be interpreted withcaution because of the difficulties in solubilizing D113 and itsanalogs.

[0271] The peptide D121 (HER-22:392-411) induced a CTL response thatlacked Ag specificity (FIG. 11A). However, D121 stimulated PBMC fromdonor 20 showed somewhat higher lysis of targets pulsed with thenonapeptide D119 than D121, but this response was short-lived. PBMC fromtwo other donors (25 and 30) stimulated with D121 and D119, were used aseffectors to determine the specificity of D119 recognition of everypeptide. Similar results were obtained with peptide stimulated PBMC fromdonor 30. Peptide recognition was also determined in 20 h cytotoxicityassays. No major differences in recognition of targets pulsed withpeptides used as stimulator versus control peptides were observed.Similarly D119 was found to increase the reactivity of MA2.1 mAb withHLA-A2 on T2 cells but failed to induce peptide specific CTLs (FIG. 11Band FIG. 11C).

EXAMPLE 3 Synthesis of Novel Universal Immunodominant Peptide Epitopes

[0272] A large number of nonapeptides (synthetic analogs) have beenconstructed, and it has been determined which ones are recognized byCTLs associated with and lysing ovarian tumors. Of more than peptidestested for recognition by three HLA-A2+ CTL lines, the followingpeptides have been recognized more often. Based on the levels of lysisinduced they were designated as high: C85 (2/3); E90 (2/3), E75 (2/3)E71 (2/3), E89 (2/3); and moderate E77 (2/3).

[0273] The sequences of these peptides are as follows: C85 =HER-2:971-979 - E L V S E F S R M (SEQ ID NO:7) E89 = HER-2:851-859 - VL V K S P N H V (SEQ ID NO:8) E71 = HER-2:798-806 - Q L M P Y G C L L(SEQ ID NO:9) E90 = HER-2:788-796 - C L T S T V Q L V (SEQ ID NO:10) E75= HER-2:370-378 - K I F G S L A F L (SEQ ID NO:11) E77 = HER-2:391-399 -P L Q P E Q L Q V (SEQ ID NO:12)

[0274] The ability of these peptides to sensitize targets for lysis bytumor associated CTLs (relative to positive control C85) is shown inTable 7.

[0275] These sequences, being immunodominant, can provide universalHER-2 targets and antigens for CTLs in the HLA-A2 system expressed byover 450 of North American population.

[0276] Since HER-2 is a self-antigen, during thymic selection, a numberof T-cells carrying receptors with high affinity for the HLA-peptidecomplex are silenced either by elimination ro finer tolerization. Apre-condition for induction of a high affinity TCR-(MHC+peptide)interactions, is a stable (MHC+peptide) complex. Therefore T-cellsreacting with peptides that bind HLA with low affinity and have weakstabilizing effect, are not likely to be eliminated in vivo but they canbecome CTL targets. However, stabilization of HLA-Class I binding byexogenously added peptide is dependent on introduction of dominantanchors in positions P2 and P9 which are not recognized by TCR. Inaddition to patenting this concept we found that replacement of Met (P9)stabilize HLA-A2 expression on an indicator line T2 used for these typesof studies. TABLE 7 RECOGNITION OF HER-2 PEPTIDES BY OVARIAN TUMORASSOCIATED CYTOTOXIC T LYMPHOCYTES CTL-24 % of C85^(a) CTL-34 % of C85High High C85^(b) 1.000 C85 1.000 E90 0.885 E90 1.149 E75 0.850 E891.149 Moderate Moderate E77 0.759 E71 0.600 E89 0.734 E77 0.300 E710.625 Negative CTL-16 D113 0.095 D99 0.050 High D97 −0.025 E75>10.00^(c)

[0277] C85=ELVSEFSRM (SEQ ID NO: 7) is the natural nonapeptiderecognized by CTL. Peptides C84=ELVSEFSRV, (SEQ ID NO: 6) andC83=ELVSEFSKV (SEQ ID NO: 5) are analogs with strengthened P9 and P8.C84 also can specifically inhibit tumor lysis by peptide induced CTL.Furthermore, Leu (P2) is a dominant anchor, but E (P1) may beelectrostatically rejected by residues that form the MHC class I bindingpocket. Thus replacement of E→G (P1)(neutral) or E→K (P1, positivecharge) are also expected to stabilize the interaction, while theresidues being buried in the pocket, are expected not to affect CTLrecognition.

[0278] The analogs with sequences C91=GLVSEFSRV, (SEQ ID NO: 4) and C92=KLVSEFSRV (SEQ ID NO: 3) are also compositions of the presentinvention. In addition, substitutions at P4 (S→K) and P6 (F→V) affectresidues that are expected to interact with TCR. The analogC81=ELVSEVSKV (SEQ ID NO: 2) stabilized HLA-A2 more than C84, while C82:ELVKEVSKV (SEQ ID NO: 1) although binds HLA-A2 is no longer recognizedby C84 reactive CTL. Both C81 and C82 can form the core for antagonistsof HER-2 reactive CTLs (to control and stop CTL reactions), and as suchrepresent the first “universal” antagonists reported for stimulatingCTLs.

[0279] Peptide D113, HLYQGCQVV (SEQ ID NO: 17), is the naturalnonapeptide HER-2:42-51. D113 stabilizes HLA-A2 on indicator T2 cells.The novel synthetic peptide analog, D114, RLLYQGCQVV (SEQ ID NO: 18),shows little improvement on stabilization of HLA-A2, but the novelpeptide, D115, GLYQGCQW (SEQ ID NO: 19), shows significantly higherimprovement which confirmed the predictions above.

EXAMPLE 4 Peptide Formulations

[0280] Peptides containing the epitope motifs described herein arecontemplated for use in therapeutics to provide universal HER-2 targetsand antigens for CTLs in the HLA-A2 system expressed by over 45% of theNorth American population. The development of therapeutics based onthese novel sequences provides induction of tumor reactive immune cellsin vivo through the formulation of synthetic cancer vaccines, as well asinduction of tumor-reactive T-cells in vitro through eitherpeptide-mediated (e.g., lipopeptide) or cell-mediated (e.g., EBV-B linesusing either autologous or HLA-A2 transfectants where the gene for thepeptide of interest is introduced, and the peptide is expressedassociated with HLA-A2 on the surface). The use of these novel peptidesas components of vaccines to prevent, or lessen the chance of cancerprogression is also contemplated.

[0281] The peptides contemplated for use, being smaller than othercompositions, such as envelope proteins, will have improvedbioavailability and half lives. If desired, stability examinations maybe performed on the peptides, including, e.g., pre-incubation in humanserum and plasma; treatment with various proteases; and alsotemperature- and pH-stability analyses. If found to be necessary, thestability of the synthetic peptides may be enhanced by any one of avariety of methods such as, for example, employing D-amino acids inplace of L-amino acids for peptide synthesis; using blocking groups liket-boc and the like; or encapsulating the peptides within liposomes. Thebio-availability of select mixtures of peptides may also be determinedby injecting radio-labeled peptides into experimental animals, such asmice and/or Rhesus monkeys, and subsequently analyzing their tissuedistribution.

[0282] If stability enhancement was desired, it is contemplated that theuse of dextrorotary amino acids (D-amino acids) would be advantageous asthis would result in even longer bioavailability due to the inability ofproteases to attack these types of structures. The peptides of thepresent invention may also be further stabilized, for example, by theaddition of groups to the N- or C-termini, such as by acylation oramination. If desired, the peptides could even be in the form oflipid-tailed peptides, formulated into surfactant-like micelles, orother peptide multimers. The preparation of peptide multimers andsurfactant-like micelles is described in detail in U.S. Ser. No.07/945,865, incorporated herein by reference. The compositions of thepresent invention are contemplated to be particularly advantageous foruse in economical and safe anti-tumor/anti-cancer therapeutics, andspecific therapeutic formulations may be tested in experimental animalmodels, such as mice, rats, rabbits, guinea pigs, cats, goats, Rhesusmonkeys, chimpanzees, and the like, in order to determine more preciselythe dosage forms required.

[0283] In addition to the peptidyl compounds described herein, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the peptide structure andthat such compounds may also be used in the same manner as the peptidesof the invention. This may be achieved by the techniques of modellingand chemical design known to those of skill in the art. For example,esterification and other alkylations may be employed to modify theterminus of a peptide to mimic a particular terminal motif structure. Itwill be understood that all such sterically similar constructs fallwithin the scope of the present invention.

[0284] Therapeutic or pharmacological compositions of the presentinvention will generally comprise an effective amount of aCTL-stimulating peptide or peptides, dissolved or dispersed in apharmaceutically acceptable medium. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic, toxic, or otherwise adverse reaction whenadministered to a human. Pharmaceutically acceptable media or carriersinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated.

[0285] Supplementary active ingredients can also be incorporated intothe therapeutic compositions of the present invention. For example, thestimulatory peptides may also be combined with peptides includingcytotoxic T-cell- or T-helper-cell-inducing epitopes (as disclosed inU.S. Ser. No. 07/945,865; incorporated herein by reference) to createpeptide cocktails for immunization and treatment.

[0286] The preparation of pharmaceutical or pharmacological compositionscontaining a CTL-stimulating peptide or peptides, includingdextrorotatory peptides, as active ingredients will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions may be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection; as tablets or other solids for oraladministration; as time release capsules; or in any other form currentlyused, including cremes, lotions, mouthwashes, inhalents and the like.

[0287] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0288] Sterile solutions suitable for intravenous administration arepreferred in certain embodiments and are contemplated to be particularlyeffective in stimulating CTLs and/or producing an immune response in ananimal. The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

[0289] A peptide or peptides can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts (formed with the free amino groups of the peptide)and which are formed with inorganic acids such as, e.g., hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine, and thelike.

[0290] The carrier can also be a solvent or dispersion mediumcontaining, e.g., water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained by inter alia the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought inter alia by various antibacterial adantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, e.g., sugars or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

[0291] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0292] The preparation of more- or highly-concentrated solutions forintramuscular injection is also contemplated. This is envisioned to haveparticular utility in facilitating the treatment of needle stickinjuries to animals or even humans. In this regard, the use of DMSO assolvent is preferred as this will result in extremely rapid penetration,delivering high concentrations of the active peptide, peptides or agentsto a small area.

[0293] The use of sterile formulations, such as saline-based washes, byveterinarians, technicians, surgeons, physicians or health care workersto cleanse a particular area in the operating field may also beparticularly useful. Therapeutic formulations in accordance with thepresent invention may also be reconstituted in the form of mouthwashes,including the peptides alone, or in conjunction with antifungalreagents. Inhalant forms are also envisioned, which again, may containactive peptides or agents alone, or in conjunction with other agents,such as, e.g., pentamidine. The therapeutic formulations of theinvention may also be prepared in forms suitable for topicaladministration, such as in cremes and lotions.

[0294] Buffered ophthalmic solutions also fall within the scope of theinvention, and may be created in accordance with conventionalpharmaceutical practice, see for example “Remington's PharmaceuticalSciences” 15th Edition, pages 1488 to 1501 (Mack Publishing Co., Easton,Pa.). Suitable ophthalmic preparations will generally contain a noveldipeptide, peptide or agent as disclosed herein in a concentration fromabout 0.01 to about 1% by weight, and preferably from about 0.05 toabout 0.5%, in a pharmaceutically acceptable solution, suspension orointment. The ophthalmic preparation will preferably be in the form of asterile buffered solution containing, if desired, additionalingredients, for example preservatives, buffers, tonicity agents,antioxidants and stabilizers, nonionic wetting or clarifying agents,viscosity-increasing agents and the like.

[0295] Suitable preservatives for use in such a solution includebenzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosaland the like. Suitable buffers include boric acid, sodium and potassiumbicarbonate, sodium and potassium borates, sodium and potassiumcarbonate, sodium acetate, sodium biphosphate and the like, in amountssufficient to maintain the pH at between about pH 6 and pH 8, andpreferably, between about pH 7 and pH 7.5. Suitable tonicity agents aredextran 40, dextran 70, dextrose, glycerin, potassium chloride,propylene glycol, sodium chloride, and the like, such that the sodiumchloride equivalent of the ophthalmic solution is in the range 0.9±0.2%.Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfate, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethyl-propylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

[0296] Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. As used herein, “pharmacologically effective amount” means anamount of composition is used that contains an amount of a peptide orpeptides sufficient to significantly stimulate a CTL or generate animmune response in an animal.

[0297] In this context, the quantity of peptide(s) and volume ofcomposition to be administered depends on the host animal to be treated,such as, the capacity of the host animal's immune system to produce animmune response. Precise amounts of active peptide required to beadministered depend on the judgment of the practitioner and are peculiarto each individual.

[0298] A minimal volume of a composition required to disperse thepeptide is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals. For example, for parenteral administration,a suitably buffered, and if necessary, isotonic aqueous solution wouldbe prepared and used for intravenous, intramuscular, subcutaneous oreven intraperitoneal administration. One dosage could be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580).

[0299] In certain embodiments, active compounds may be administeredorally. This is contemplated for agents that are generally resistant, orhave been rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include chemically designed or modifiedagents; dextrorotatory peptides; and peptide and liposomal formulationsin timed-release capsules to avoid peptidase, protease and/or lipasedegradation.

[0300] Oral formulations may include compounds in combination with aninert diluent or an edible carrier which may be assimilated; thoseenclosed in hard- or soft-shell gelatin capsules; those compressed intotablets; or those incorporated directly with the food of the diet. Fororal therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should generallycontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60%. of the weight of the unit.The amount of active compounds in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

[0301] Tablets, troches, pills, capsules and the like may also containthe following: a binder, as gum tragacanth, acacia, corn starch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent methyl and propylparaben as preservatives,a dye and flavoring, such as cherry or orange flavor. Of course, anymaterial used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0302] The peptides may be used in their immunizing capacity byadministering an amount effective to generate an immune response in ananimal. In this sense, such an “amount effective to generate an immuneresponse” means an amount of composition that contains a peptide orpeptide mixture sufficient to significantly produce an antigenicresponse in said animal.

EXAMPLE 5 Methods for Protein Size Determination and Gel Chromatography

[0303] The amino acid sequences disclosed herein, and particularly thetripeptide motifs and multimers thereof, find particular use in thedetermination of molecular weights of small polypeptides. These peptidesrepresent a significant improvement over commercially-available proteinstandards in this area owing to their small size and, since their aminoacid sequence is known, their precise molecular weight is readilydetermined.

[0304] 1. SDS-PAGE Analysis of Proteins

[0305] Commercially-available protein standards for SDS-PAGE or gelfiltration chromatography typically have a range of 3,000 to 200,000 Da(Gibco BRL, Bethesda, Md.), and as such, are not useful in thecharacterization of proteins having molecular weights of about 300 toabout 3,000 Da. By employing peptides of the present invention (e.g.,SEQ ID NOS: 1-15) and multimers thereof, a range of suitablelow-molecular weight standards may be readily prepared. Such a molecularweight ladder mixture may be employed either in SDS-PAGE or gelfiltration protocols which are well-known to those of skill in the art(see e.g., Wood, 1981).

[0306] 2. Paper and Thin-Layer Chromatography

[0307] In a similar fashion, the polypeptides, and more particularly thetripeptide motifs, of the present invention are readily employed asstandards in the identification of small molecular-weight polypeptidesusing chromatographic separation. In preferred embodiments, paperchromatography is utilized and proteins are subsequently visualizedafter reaction with ninhydrin. More preferred is the use of thin-layerchromatography in either one or two dimensions.

[0308] 3. Gel Filtration Chromatography

[0309] The polypeptides of the present invention provide excellentstandards for the calibration of chromatographic columns used in theseparation of low molecular-weight polypeptides. In particular, thetripeptide motifs, and multimers thereof, find important use in thestandardization of low-molecular weight-range columns (Rawn, 1983).These chromatography columns may include a filtration medium having thecapacity to fractionate any protein of interest and the polypeptides ofthe present invention. Chromatographic media such as G-50 or G-25Sephadex® resins (approximate fractionation range of 1,500-30,000 and100-5,000 Da, respectively) may be used for generalized separation, orin cases where the approximate molecular weight of the protein ofinterest is known, a medium having a narrower fractionation range (e.g.,G-10 Sephadex® [0-700 Da separation range] or G-15 Sephadex® [0-1,500 Daseparation range]) may be employed. A regression line of the elutionposition versus the log of the molecular weight is established using thepeptides of the present invention, and the molecular weight of theprotein of interest is then determined from this graph. Detailedprotocols for preparation, calibration, and execution of these columnsis well-known to those of skill in the art (see e.g., Wood, 1981).

[0310] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe composition, methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

[0311] References

[0312] The following literature citations as well as those cited aboveare incorporated in pertinent part by reference herein for the reasonscited in the above text.

[0313] Aebersold et al., “Lysis of autologous melanoma cells bytumor-infiltrating lymphocytes: association with clinical response,” J.Natl. Cancer Inst., 83:932-937, 1991.

[0314] Alexander et al., “Correlation between CD8 dependency anddeterminant density using peptide-induced, Ld-restricted cytotoxic Tlymphocytes,” J. Exp. Med., 173:849-858, 1991.

[0315] Anderson et al., “Intracellular transport of Class I MHCmolecules in antigen processing mutant cell lines,” J. Immunol.,151:3407-3419, 1993.

[0316] Anderson et al., Mol. Immunol., 29:1089, 1992.

[0317] Ausubel, F. M. et al., “Current Protocols in Molecular Biology,”John Wiley & Sons, New York, 1989.

[0318] Bednarek et al., “The minimum peptide epitope from the influenzavirus matrix protein: extra and intracellular loading of HLA-A2,” J.Immunol., 147:4047-4053, 1991.

[0319] Bednareket al., “Soluble HLA-A2.1 restricted peptides that arerecognized by influenza virus specific cytotoxic T lymphocytes,” J.Immunol. Meth., 139:41-47, 1991.

[0320] Bowness et al., Eur. J. Immunol., 23:1417, 1993.

[0321] Brichard et al., J. Exp. Med., 178:489, 1993.

[0322] Brock et al., “Biology of Microorganisms” 7th Edition, PrenticeHall, Inc., Englewood Cliffs, N.J., 1994.

[0323] Campbell, “Monoclonal Antibody Technology, Laboratory Techniquesin Biochemistry and Molecular Biology,” Vol. 13, Burden and VonKnippenberg, Eds. pp. 75-83, Elsevier, Amsterdam, 1984.

[0324] Carbone et al., “Induction of cytotoxic T lymphocytes by primaryin vitro stimulation with peptides,” J. Exp. Med., 167:1767-1779, 1988.

[0325] Coakley and James, “A Simple Linear Transform for the Folin-LowryProtein Calibration Curve to 1.0 mg/ml,” Anal. Biochem. 35. 85:90-97,1978.

[0326] DeLisi and Berzofsky, “T-cell antigenic sites tend to beamphipathic structures,” Proc. Natl. Acad. Sci. USA, 82:7048-7052, 1985.

[0327] Dibrino et al., J. Immunol., 152:620, 1994.

[0328] Falk et al., “Allele-specific motifs revealed by sequencing ofself-peptides eluted from MHC molecules,” Nature, 351:290-45 296, 1991.

[0329] Fisk et al., “Oligopeptide induction of a cytotoxic T lymphocyteresponse to HER-2/Neu proto-oncogene in vitro,” Cell. Immunol.157:415-427, 1994a.

[0330] Fisk et al., “Sequence motifs of human HER-2 proto-oncogeneimportant for peptide binding to HLA-A2,” Int. J. Oncol. 5:51-63, 1994b.

[0331] Fisk et al., “Oligopeptide induction of a cytotoxic T lymphocyteresponse to HER-2/neu proto-oncogene product in vitro,” Proc. AACR,35:498, 1994c.

[0332] Gammon et al., “Endogenous loading of HLA-A2 molecules with ananalog of the influenza virus matrix protein-derived peptide and itsinhibition by an exogenous peptide antagonist,” J. Immunol., 148:7-12,1992.

[0333] Gefter et al., Somatic Cell Genet. 3:231-236, 1977.

[0334] Gendler et al., J. Biol. Chem., 263:12820, 1988.

[0335] Goding, “Monoclonal Antibodies: Principles and Practice,” pp.60-74. 2nd Edition, Academic Press, Orlando, Fla., 1986.

[0336] Grimm et al., J. Exp. Med., 155:1823, 1982.

[0337] Harlow, E. and Lane, D. “Antibodies: A Laboratory Manual,” ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.

[0338] Hill et al., Nature, 360:434, 1992.

[0339] Hogquist et al., “Peptide variants reveal how antibodiesrecognize major histocompatibility complex class I,” Eur. J. Immunol.,23:3028-3036, 1993.

[0340] Houbiers et al., “In vitro induction of human cytotoxic Tlymphocyte responses against peptides of mutant and wild-type p53,” Eur.J. Immunol., 23:2072-2077, 1993.

[0341] Hu et al., J. Exp. Med., 177:1681, 1993.

[0342] Ioannides et al., “Cytotoxic T-cell clones isolated from ovariantumor-infiltrating lymphocytes recognize multiple antigenic epitopes onautologous tumor cells,” J. Immunol., 146:1700-1707, 1991.

[0343] Ioannides et al., “Cytotoxic T-cells isolated from ovarianmalignant ascites recognize a peptide derived from the HER-2/neu protoonco gene,” Cell Immunol., 136:225-234, 1993.

[0344] Ioannides et al., “T-cell recognition of oncogene products,” Mol.Carcinogen, 6:77-81, 1992.

[0345] Ioannides et al., “Tumor cytolysis by lymphocytes infiltratingovarian malignant ascites,” Cancer Res., 51:4257-4264, 1991.

[0346] Ioannides et al., Scand. J. Immunol., 37:413, 1993.

[0347] Jacobson et al., J. Virol., 63:1756, 1989.

[0348] Jameson & Wolf, Compu. Appl. Biosci., 4(1):181-6, 1988.

[0349] Jerome et al., Cancer Res., 51:2908, 1991.

[0350] Jerome et al., J. Immunol., 151:1654, 1993.

[0351] Kaiser and Kezdy, “Amphiphilic secondary structure: Design ofpeptide hormones,” Science, 223:249-255, 1984.

[0352] Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976.

[0353] Kohler and Milstein, Nature 256:495-497, 1975.

[0354] Kos and Müllbacher, Eur. J. Immunol., 22:3183, 1992.

[0355] Kyte and Doolittle, 1982

[0356] Letessier et al., Cancer Res., 51:3891, 1991.

[0357] Lowry et al., “Protein Measurement with the Folin PhenolReagent,” J. Biol. Chem. 193:265-275, 1951.

[0358] Madden et al., “The antigenic identity of peptide-MHC complexes:a comparison of the conformations of five viral peptides presented byHLA-A2,” Cell, 75:693-708, 1993.

[0359] Maloy, S. R., “Experimental Techniques in Bacterial Genetics”Jones and Bartlett Publishers, Boston, Mass., 1990.

[0360] Maloy et al., “Microbial Genetics” 2nd Edition. Jones and BarlettPublishers, Boston, Mass., 1994.

[0361] Marincola et al., Cancer Res., 83:932, 1991.

[0362] Mullis, K., U.S. Pat. No. 4,683,202, Jul. 28, 1987.

[0363] Mullis, K. et al., U.S. Pat. No. 4,683,195, Jul. 28, 1987.

[0364] Ohno, Proc. Natl. Acad. Sci. USA, 88:3065, 1991.

[0365] Parker et al., “Sequence motifs important for peptide binding tothe human MHC class I molecule, HLA-A2,” J. Immunol., 149:3580-3587,1992.

[0366] Parmiani, “Tumor immunity as autoimmunity: tumor antigens includenormal self proteins which stimulate anergic peripheral T-cells,”Immunol. Today, 14:536-538, 1993.

[0367] Prokop, A., and Bajpai, R. K. “Recombinant DNA Technology I” Ann.N. Y. Acad. Sci. vol. 646, 1991.

[0368] Ratner and Clark, J. Immunol., 150:4303, 1993.

[0369] Rawn, J. D. “Biochemistry” Harper & Row Publishers, New York,1983.

[0370] Rosenberg et al., “Use of tumor-infiltrating lymphocytes andinterleukin-2 in the immunotherapy of patients with metastaticmelanoma,” N. Engl. J. Med., 319:1676-1680, 1988.

[0371] Rothbard and Taylor, “A sequence common to T-cell epitopes,” EMBOJ., 7:93-100, 1988.

[0372] Salter and Creswell, “Impaired assembly and transport of HLA-Aand -B antigens in a mutant T×B cell hybrid,” EMBO J., 5:943-949, 1986.

[0373] Salter et al., “IN vitro mutagenesis at a single residueintroduces B- and T-cell epitopes into a class I HLA molecule,” J. Exp.Med., 166:283-288, 1987.

[0374] Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

[0375] Santos-Aguado et al., “Molecular characterization of serologicrecognition sites in the human HLA-A2 molecule,” J. Immunol.,141:2811-2818, 1988.

[0376] Saper et al., “Refined structure of the human histocompatibilityantigen HLA-A2 at 2, 6 Å resolution,” J. Mol. Biol., 219:277-319, 1991.

[0377] Schild et al., “Fine specificity of cytotoxic T lymphocytesprimed in vivo either with virus or synthetic lipopeptide vaccine orprimed in vitro with peptide,” J. Exp. Med., 174:1665-1668, 1991.

[0378] Schmidt et al., “Oligopeptide induction of a secondary cytotoxicT-cell response to Epstein-Barr virus in vitro,” Scand. J. Immunol.,33:411-420, 1991.

[0379] Segal, I. H., “Biochemical Calculations” 2nd Edition. John Wiley& Sons, New York, 1976.

[0380] Sherman et al., J. Exp. Med., 175:1221, 1992.

[0381] Slamon et al., “Studies of the HER-2/neu proto-oncogene in humanbreast and ovarian cancer,” Science, 244:707-712, 1989.

[0382] Stauss et al., “Induction of cytotoxic T lymphocytes withpeptides in vitro: Identification of candidate,T-cell epitopes in humanpapilloma virus,” Proc. Natl. Acad. Sci. USA, 89:7871-7875, 1992.

[0383] Steinman, Annu. Rev. Immunol., 9:271, 1991.

[0384] Suhrbier et al., J. Immunol., 150:2169, 1993.

[0385] Walden and Eisen, Proc. Natl. Acad. Sci. USA, 87:9015, 1990.

[0386] Winter et al., J. Immunol., 146:3508, 1991.

[0387] Wolf et al., Compu. Appl. Biosci., 4(1):187-91 1988.

[0388] Wolfel et al., Int. J. Cancer, 54:636, 1993.

[0389] Wood, R. A., “Metabolism,” In Manual of Methods for GeneralBacteriology, (Gerhardt, Murray, Costilow, Nester, Wood, Krieg, andPhillips, Eds.) American Society for Microbiology, Washington, D.C.,1981.

[0390] Yamamoto et al., “Similarity of protein encoded by the humanc-erb-B-2 gene to epidermal growth factor receptor,” Nature,319:230-234, 1986.

[0391] Young and Davis, “Efficient Isolation of Genes by Using AntibodyProbes,” Proc. Natl. Acad. Sci. USA, 80:1194-1198, 1983.

[0392] Zweerink et al., “Presentation of endogenous peptides to MHCclass I-restricted cytotoxic T lymphocytes in transport deletion mutantT2 cells,” J. Immunol., 150:1763-1771, 1993.

1 68 1 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 1 Glu Leu Val Lys Glu Val Ser Lys Val 1 5 2 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide2 Glu Leu Val Ser Glu Val Ser Lys Val 1 5 3 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 3 Lys Leu Val SerGlu Phe Ser Arg Val 1 5 4 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 4 Gly Leu Val Ser Glu Phe Ser ArgVal 1 5 5 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 5 Glu Leu Val Ser Glu Phe Ser Lys Val 1 5 6 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide6 Glu Leu Val Ser Glu Phe Ser Arg Val 1 5 7 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 7 Glu Leu Val SerGlu Phe Ser Arg Met 1 5 8 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 8 Val Leu Val Lys Ser Pro Asn HisVal 1 5 9 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 9 Gln Leu Met Pro Tyr Gly Cys Leu Leu 1 5 10 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide10 Cys Leu Thr Ser Thr Val Gln Leu Val 1 5 11 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 11 Lys Ile Phe GlySer Leu Ala Phe Leu 1 5 12 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 12 Pro Leu Gln Pro Glu Gln Leu GlnVal 1 5 13 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 13 Thr Leu Glu Glu Ile Thr Gly Tyr Leu 1 5 14 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide14 Gln Leu Gln Val Phe Glu Thr Leu Glu 1 5 15 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 15 Gly Leu Gly MetGlu His Leu Arg Glu Val 1 5 10 16 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic Peptide 16 Asp Leu Gly Met Gly Ala AlaLys Gly Leu 1 5 10 17 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 17 His Leu Tyr Gln Gly Cys Gln ValVal 1 5 18 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 18 Arg Leu Leu Tyr Gln Gly Cys Gln Val Val 1 5 10 19 9PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 19 Gly Leu Tyr Gln Gly Cys Gln Val Val 1 5 20 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 20 Asp IleGln Glu Val Gln Gly Tyr Val 1 5 21 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic Peptide 21 Gln Leu Arg Ser Leu Thr GluIle Leu 1 5 22 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 22 Asp Ile Phe His Lys Asn Asn Gln Leu 1 5 239 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 23 Tyr Ile Ser Ala Trp Pro Asp Ser Leu 1 5 24 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 24 Ser LeuArg Glu Leu Gly Ser Gly Leu 1 5 25 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic Peptide 25 Pro Leu Thr Ser Ile Ile SerAla Val 1 5 26 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 26 Arg Leu Leu Gln Glu Thr Glu Leu Val 1 5 279 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 27 Lys Ile Pro Val Ala Ile Lys Val Leu 1 5 28 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 28 Gln IleAla Lys Gly Met Ser Tyr Leu 1 5 29 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic Peptide 29 Thr Leu Ser Pro Gly Lys AsnGly Val 1 5 30 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 30 His Leu Asp Met Leu Arg His Leu Tyr Gln 15 10 31 14 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 31 Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly CysGln 1 5 10 32 12 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 32 Asn Gln Glu Val Thr Ala Trp Asp Gly ThrGln Arg 1 5 10 33 14 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 33 Leu Gln Val Phe Glu Thr Leu Glu Glu IleThr Gly Tyr Leu 1 5 10 34 20 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 34 Leu Gln Pro Glu Gln Leu Gln ValPhe Glu Thr Leu Glu Glu Ile Thr 1 5 10 15 Gly Tyr Leu Tyr 20 35 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide35 Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 1 5 10 36 13 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide36 Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu 1 5 10 37 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide37 Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg 1 5 10 38 14 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide38 Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg 1 5 10 39 14PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 39 Arg Phe Arg Glu Leu Ile Ile Glu Phe Ser Arg Met Ala Arg 1 510 40 13 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 40 Leu Val Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln1 5 10 41 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 41 Arg Phe Arg Glu Leu Val Ser Glu Phe Ser 1 5 10 42 9PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 42 Glu Cys Arg Pro Arg Phe Arg Glu Leu 1 5 43 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 43 Arg PheArg Glu Leu Val Ser 1 5 44 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 44 Asp Leu Gly Met Gly Ala Ala LysGly Leu 1 5 10 45 13 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 45 Phe Asp Gly Asp Leu Gly Met Gly Ala AlaLys Gly Leu 1 5 10 46 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 46 Arg Ile Ala Trp Ala Arg Thr GluLeu 1 5 47 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 47 Asn Leu Gly Pro Trp Ile Gln Gln Val 1 5 48 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide48 Glu Ile Trp Thr His Ser Thr Lys Val 1 5 49 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 49 Ser Leu Ala LeuMet Leu Leu Trp Leu 1 5 50 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 50 Leu Leu Ser Leu Ala Leu Met LeuLeu 1 5 51 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 51 gaaytngtna argaagtnws naargtn 27 52 27 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer52 gaaytngtnw sngaagtnws naargtn 27 53 27 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 53 aarytngtnwsngaattyws nmgngtn 27 54 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 54 ggnytngtnw sngaattyws nmgngtn 2755 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 55 gaaytngtnw sngaattyws naargtn 27 56 27 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer56 gaaytngtnw sngaattyws nmgngtn 27 57 27 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 57 gaaytngtnwsngaattyws nmgnatg 27 58 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 58 gtnytngtna arwsnccnaa ycaygtn 2759 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 59 carytnatgc cntaygartg yytnytn 27 60 27 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer60 tgyytnacnw snacngtnca rytngtn 27 61 27 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 61 aarathttyggnwsnytngc nttyytn 27 62 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 62 ccnytncarc cngarcaryt ncargtn 2763 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 63 acnytngarg arathacngg ntayytn 27 64 24 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer64 carytncarg tnttygarac nytn 24 65 14 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 65 Arg Phe Arg GluLeu Val Ser Glu Phe Ser Arg Met Ala Arg 1 5 10 66 14 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 66 Arg PheArg Glu Leu Ile Ile Glu Phe Ser Arg Met Ala Arg 1 5 10 67 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide67 Ser Leu Ala Asp Pro Ala His Gly Val 1 5 68 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 68 Gly Leu Thr SerAla Pro Asp Thr Arg Val 1 5 10

What is claimed is:
 1. A peptide of between 8 and about 20 amino acid residues in length, and including within its sequence the amino acid sequence of: AA₁—AA₂—AA₃—AA₄—AA₅—AA₆—AA₇—AA₈; wherein AA₁ is Leu or Ile; AA₂ is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; AA₃ is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val; AA₄ is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val; AA₅ is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val; AA₆ is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr; AA₇ is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA₈ is Val, Leu, Met, Gly, or Glu.
 2. The peptide of claim 1, further defined as a peptide of between 8 and about 15 amino acid residues in length.
 3. The peptide of claim 2, further defined as a peptide of between 8 and 10 amino acid residues in length.
 4. The peptide of claim 3, further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO:
 29. 5. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 1. 6. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 2. 7. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 3. 8. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 4. 9. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 5. 10. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 6. 11. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 7. 12. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 8. 13. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 9. 14. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 18. 15. The peptide of claim 4, further defined as having the amino acid sequence of SEQ ID NO:
 19. 16. The peptide of claim 1, wherein AA₂ is Val or Gln; AA₃ is Ser, Gln, Glu, Lys, or Pro; AA₄ is Glu, Gly, Ile, Leu, or Ser; AA₆ is Asn, Gln, or Ser; and AA₇ is Arg, Leu, Gln, Tyr, Val, or Lys.
 17. The peptide of claim 16, wherein AA₂ i s Val; AA₃ is Ser; AA₄ is Glu; AA₆ is Ser; and AA₇ is Arg or Lys.
 18. A peptide of between 8 and about 20 amino acid residues in length, said peptide stimulating cytotoxic T-lymphocytes and comprising the amino acid sequence: AA₁—AA₂—AA₃—AA₄—AA₅—AA₆—AA₇—AA₈; wherein AA₁ is Leu or Ile; AA₂ is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; AA₃ is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val; AA₄ is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val; AA₅ is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val; AA₆ is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr; AA₇ is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA₈ is Val, Leu, Met, Gly, or Glu.
 19. The peptide of claim 18, further defined as being from 8 amino acid residues in length to about 15 amino acid residues in length.
 20. The peptide of claim 19, further defined as being from 8 amino acid residues in length to about 10 amino acid residues in length.
 21. The peptide of claim 20, further defined as being 8 amino acid residues in length.
 22. The peptide of claim 21, further defined as being 9 amino acid residues in length.
 23. The peptide of claim 21, further defined as being 10 amino acid residues in length.
 24. The peptide of claim 18, further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO:
 29. 25. The peptide of claim 24, further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, or SEQ ID NO:
 19. 26. A peptide of between 8 and about 20 amino acid residues in length, said peptide binding HLA and stimulating cytotoxic T-lymphocytes, and including within its sequence an amino acid sequence represented by: AA₁—AA₂—AA₃—AA₄—AA₅—AA₆—AA₇—AA₈; wherein AA₁ is Leu or Ile; AA₂ is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; AA₃ is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val; AA₄ is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val; AA₅ is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val; AA₆ is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr; AA₇ is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA₈ is Val, Leu, Met, Gly, or Glu.
 27. The peptide of claim 26, wherein AA₂ is Val or Gln; AA₃ is Ser, Gln, Glu, Lys, or Pro; AA₄ is Glu, Gly, Ile, Leu, or Ser; AA₆ is Asn, Gln, or Ser; and AA₇ is Arg, Leu, Gln, Tyr, Val, or Lys.
 28. The peptide of claim 27, wherein AA₂ is Val; AA₃ is Ser; AA₄ is Glu; AA₆ is Ser; and AA₇ is Arg or Lys.
 29. The peptide of claim 26, further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO:
 29. 30. A method for stimulating cytotoxic T-lymphocytes, comprising contacting said cytotoxic T-lymphocytes with an amount of a peptide in accordance with claim 1 effective to stimulate said cytotoxic T-lymphocytes.
 31. The method of claim 30, wherein said cytotoxic T-lymphocytes are located within an animal and said peptide or composition is administered to said animal.
 32. The method of claim 30, wherein said cytotoxic T-lymphocytes are obtained from an animal, contacted with said peptide, and re-administered to said animal.
 33. The method of claim 30, wherein said peptide is formulated for administration parenterally, topically, or as an inhalant, aerosol or spray.
 34. The method of claim 31, wherein said animal is a human subject.
 35. A pharmaceutical composition including the composition of claim 1 in a pharmaceutically acceptable excipient.
 36. The pharmaceutical composition of claim 35, wherein said composition comprises the peptide of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO:
 29. 37. A method of treating a proliferative cell disorder in an animal, comprising administering to said animal a therapeutically-effective amount of a pharmaceutical composition in accordance with claim
 35. 38. The method of claim 37, wherein said proliferative cell disorder is cancer.
 39. The method of claim 38, wherein said cancer is breast or ovarian cancer.
 40. A method for detecting cytotoxic T-lymphocytes in a sample, comprising obtaining a sample suspected of containing cytotoxic T-lymphocytes, contacting said sample with a peptide in accordance with claim 1, under conditions effective to allow the formation of cell-peptide complexes, and detecting the cell-peptide complexes so formed.
 41. The method of claim 40, wherein said sample is a biological sample from an animal suspected of having a HER-2/neu-related cancer.
 42. The method of claim 40, wherein said peptide is linked to a detectable label and the cell-peptide complexes are detected by detecting the presence of the label.
 43. The method of claim 40, wherein said cell-peptide complexes are detected by means of an antibody linked to a detectable label, the antibody having binding affinity for the peptide.
 44. A method of generating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a composition comprising the peptide of claim
 1. 45. The method of claim 44, wherein said composition comprises an immunologically effective amount of a composition comprising the peptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO:
 29. 46. A purified antibody that binds to the peptide of claim
 1. 47. The antibody of claim 46, wherein the antibody is a monoclonal antibody.
 48. The antibody of claim 46, wherein the antibody is linked to a detectable label.
 49. The antibody of claim 48, wherein the antibody is linked to a radioactive label, a fluorogenic label, a nuclear magnetic spin resonance label, biotin or an enzyme that generates a colored product upon contact with a chromogenic substrate.
 50. The antibody of claim 49, wherein the antibody is linked to an alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.
 51. A method for detecting a neu-containing cancer cell, a neu protein, or neu peptide; the method comprising: (a) generating an antibody that binds to the peptide of claim
 1. (b) obtaining a sample suspected of containing a neu-containing cancer cell, a neu protein, or neu peptide; (c) contacting said sample with said antibody, under conditions effective to allow the formation of immune complexes; and (d) detecting the immune complexes so formed.
 52. The method of claim 51, wherein said antibody is a monoclonal antibody.
 53. An immunodetection kit comprising, in suitable container means, the peptide of claim 1, or a first antibody that binds to the peptide of claim 1, and an immunodetection reagent.
 54. The immunodetection kit of claim 53, wherein the immunodetection reagent is a detectable label that is linked to said peptide or said first antibody.
 55. The immunodetection kit of claim 54, wherein the immunodetection reagent is a detectable label that is linked to a second antibody that has binding affinity for said peptide or said first antibody.
 56. The immunodetection kit of claim 54, wherein the immunodetection reagent is a detectable label that is linked to a second antibody that has binding affinity for a human antibody.
 57. A DNA segment encoding the peptide of claim
 1. 58. The DNA segment of claim 57, further defined as encoding the peptide of claim
 3. 59. The DNA segment of claim 57, further defined as encoding the peptide of claim
 4. 60. The DNA segment of claim 57, further defined as comprising the DNA sequence of SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; or SEQ ID NO:
 64. 61. A recombinant vector comprising the DNA segment of claim
 57. 62. The recombinant vector of claim 61, further defined as comprising a DNA segment encoding a peptide which stimulates a cytotoxic T-lymphocyte. 